Reaction product of n-nonanoic acid (C9), n-heptanoic acid (C7), n-pentanoic acid (C5), mixed tetraesters with pentaerythritol, and 3,5,5-trimethylhexanoic acid

Endpoint summary

Description of key information

Subchronic 90 day oral toxicity- NOAEL  ≥1000 mg/kg/day male/female Sprague Dawley rats.

Subchronic 90 day inhalation toxicity: NOAEC 0.5 mg/L air male/female Sprague Dawley rats

Subchronic 90 day dermal toxicity- NOAEL  ≥2 000 mg/kg/day male/female Sprague Dawley rats

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 June to 22 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000.

Deviations:

not specified

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany.
Age at Start of Treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within 2 20% of the sex mean.
Identification: Earmark and tattoo.

ALLOCATION
Group Dose level mg/kg/day Number of animals Animal numbers
Males Females Males Females
1 0 (vehicle) 5 5 1-5 21-25
2 50 5 5 6-10 26-30
3 150 5 5 11-15 31-35
4 1000 5 5 16-20 36-40

The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 384852).

ANIMAL HUSBANDRY
Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 18.6-23.7°C), a relative humidity of 30-70% (actual range: 41-87%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity.
Temporary fluctuations from the light/dark cycle (with a maximum of I hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type Ill, height 15 cm.) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage,
Germany). Each batch is analysed far nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water: Free access to tap water.
Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

Vehicle: Propylene glycol, specific gravity 1.036.
Rationale for vehicle: Based on trial formulations performed at NOTOX.
Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance (0.97 g/ml) and vehicle.
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of fresh formulations prepared after the in-life phase were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Since a structurally similar Hatcol test substance (HATCOL 5236) was not stable over a 4-hour period in propylene glycol, stability of HATCOL 3331 in propylene glycol was determined over a I-, 2- and 4-hour period (highest and lowest concentration). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 364938).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.

Remarks:

Doses / Concentrations:
0, 50, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

TREATMENT: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Method: Oral gavage, using a stainless steel stomach tube.
Formulations were placed on a magnetic stirrer during dosing.
Frequency: Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Positive control:

Positive control not required in this study.

Observations and examinations performed and frequency:

Mortality/Viability: At least twice daily.
Clinical signs: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter (except at the end of week 4), this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
Functional Observations: During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson 'Technical Services, Debenham, Stowmarket, England).
Body weights: On days 1, 8, 15, 22 and 28.
Food consumption: Weekly
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Sacrifice and pathology:

CLINICAL LABORATORY INVESTIGATIONS: Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post modem examination, between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (1.0 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml).
NECROPSY: All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus
HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
HISTOPATHOLOGY
The following slides were examined by a pathologist:
- all tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group;
- all gross lesions of all animals.
Based on the treatment related morphologic changes, kidneys were also examined from all male rats of the intermediate dose groups. All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.

Other examinations:

No further examinations detailed in the study report.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment.
Incidental findings that were noted included diarrhoea (one control male), watery discharge from the eye (one male at 50 mg/kg/day), scabs and/or alopecia an the neck (one male and female at 150 mg/kg/day), piloerection (two high dose males on days 8 and 9 only), and a broken tail apex and rales (one female at 150 and 1000 mg/kg/day respectively). At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted among control females and females dosed at 50 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A slightly lower weight gain was noted for high dose males on day 8 (p<0.01).
No further changes were noted regarding body weights and body weight gain of treated animals over the 4-week study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

An increased absolute and relative food intake was noted for high dose males in weeks 1 and 2.
Other treated animals showed similar food consumption before or after allowance for body weight when compared to control animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Red blood cell count (RBC) and haematocrit levels (HCT) were slightly increased in high dose females.
No dose-response relationship was observed for the lower prothrombin time (PT) in males at 50 mg/kg/day, for the increased haemoglobin (HBG) values in females at 50 and 1000 mg/kg/day, for the higher mean corpuscular haemoglobin concentration (MCHC) and partial thromboplastin time (APTT) in females at 50 mg/kg/day, and for the reduced red cell distribution width (RDW) in females at 50 mg/kg/day and above. These changes were considered to be of no toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry values were similar between control and treated rats.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with HATCOL 3331, when compared to control animals.
The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights and liver to body weight ratios of high dose females were increased.
The increased liver weight and liver to body weight ratio of one high dose male (no. 17) correlated to the macroscopic observation of enlargement (see above). This was considered to be an incidental finding. The statistically significant reduction of thymus to body weight ratio of males at 150 mg/kg/day was considered not to be a sign of toxicity in the absence of a dose response effect. The lower spleen weights and spleen to body weight ratios of females dosed at 50 mg/kg/day and above were considered to be due to slightly higher spleen weights of controls. Similarly, the lower kidney to body weight ratios of females at 150 and 1000 mg/kg/day were considered to have attained statistical significance due to slightly higher kidney weights of control animals. Also, no dose-response relationship was apparent. These changes were considered to be of no toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy did not reveal any toxicologically relevant alterations.
No microscopic correlate was found for the enlarged liver in one high dose male (no. 17). This was considered to be an incidental finding. Other incidental findings among control and/or treated animals included an accentuated lobular pattern of the liver, pelvic dilation of the kidneys, pale discolouration of the kidneys, red discolouration of the thymus, lungs or mandibular lymph nodes, enlarged mandibular lymph nodes, red foci on the thymus, fluid in the uterus, and a broken tail apex. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance. No findings were noted in control males.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were noted in the kidneys and consisted of:
- Hyaline droplets: 215 males at 50 mg/kg/day, and all males at 150 and 1000 mg/kg/day (minimal to moderate severity);
- Increased incidence and severity of tubular basophilia (slight to moderate): 315 males at 1000 mg/kg/day and 215 males at 150 mg/kg/day;
- Degeneration and/or necrosis of the tubular epithelium of the renal cortex: all males at 1000 mg/kg/day, 415 males at 150 mg/kg/day, and 115 males at 50 mg/kg/day.
All remaining microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

Analysis of Dose Preparations: Analyses were based on three peaks in the chromatogram of HATCOL 3331. These analyses revealed that test substance formulations in propylene glycol were stable for at least 4 hours (i.e. recovery after 4 hours exceeded 90% of nominal on average) and formed a homogeneous suspension at the concentrations tested. Accuracy of group 3 formulations was confirmed (i.e. between 91 and 102% of nominal). Although accuracy measurements of the other dose preparations revealed some values being slightly below the range of 90-1 10% of nominal, mean accuracy values were within the nominal range. Therefore, accuracy of formulations was considered to be acceptable for the type of formulations used in this study, also taking into account the nature of the test substance and the analytical method used.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

no

FUNCTIONAL OBSERVATIONS

MALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 mg/kg	150 mg/kg	1000 mg/kg
AT WEEK 4
HEARING	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL L	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
STATIC R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
GRIP	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 mg/kg	150 mg/kg	1000 mg/kg
AT WEEK 4
HEARING	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL L	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
STATIC R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
GRIP	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

BODYWEIGHTS (GRAM) SUMMARY

MALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 mg/kg	150 mg/kg	1000 mg/kg
TREATMENT
DAY 1	MEAN	190	188	190	191
WEEK 1	ST.DEV.	5.8	7.1	4.8	7.7
	N	5	5	5	5
DAY 8	MEAN	247	243	248	235
WEEK 2	ST.DEV.	9.0	5.2	11.0	9.2
	N	5	5	5	5
DAY 15	MEAN	293	284	296	295
WEEK 3	ST.DEV.	12.8	8.9	19.1	18.0
	N	5	5	5	5
DAY 22	MEAN	330	319	336	332
WEEK 4	ST.DEV.	13.1	12.5	27.3	23.5
	N	5	5	5	5
DAY 28	MEAN	347	334	353	351
WEEK 4	ST.DEV.	11.7	15.2	30.1	32.0
	N	5	5	5	5
FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 mg/kg	150 mg/kg	1000 mg/kg
TREATMENT
DAY 1	MEAN	154	153	154	153
WEEK 1	ST.DEV.	5.9	8.2	7.7	6.6
	N	5	5	5	5
DAY 8	MEAN	181	181	181	181
WEEK 2	ST.DEV.	8.1	7.2	4.3	8.1
	N	5	5	5	5
DAY 15	MEAN	201	203	200	204
WEEK 3	ST.DEV.	9.4	7.6	2.9	9.9
	N	5	5	5	5
DAY 22	MEAN	220	222	223	222
WEEK 4	ST.DEV.	18.4	13.5	6.3	13.4
	N	5	5	5	5

*/** Dunnett-test on pooled variance significant at 5% (*) or 1% (**) level

 

MACROSCOPIC FINDIGNS SUMMARY

MALES
	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	50 mg/kg	150 mg/kg	1000 mg/kg
END OF TREATMENT
Animals examined	5	5	5	5
Animal without findings	5	2	3	3
Animals affected	0	3	2	2
Liver
Accentuated lobular pattern	0	0	1	0
Enlarged	0	0	0	1
Kidneys
Pelvic dilation	0	2	0	0
Discolouration	0	0	1	0
Thymus
Discolouration	0	0	2	0
Mandibular l. node
Enlarged	0	2	0	1
FEMALES
	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	50 mg/kg	150 mg/kg	1000 mg/kg
END OF TREATMENT
Animals examined	5	5	5	5
Animals without findings	2	3	1	3
Animals affected	3	2	4	2
Lungs
Discolouration	0	0	1	0
Kidneys
Pelvic dilation	0	0	0	1
Uterus
Contains fluid	2	0	1	1
Thymus
Focus/foci	1	1	0	0
Mandibular l. node
Enlarged	1	1	1	0
Discolouration	0	0	1	1
Bone
Tail, apex, broken	0	0	1	0

 

Conclusions:

From the results presented in the report a No Observed Adverse Effect Level (NOAEL) could not be determined for males, but was established to be 150 mg/kg/day for females. However, since the male kidney findings are a species and sex specific response which is not observed in humans, a NOAEL of 150 mg/kg/day may be considered for both sexes.

Executive summary:

Subacute 28-day oral toxicity with HATCOL 3331 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

The study was based on the following guidelines:

-EC Directive 96/541EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996.

-OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995-OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (71 01), EPA 712-C-00-366, 2000.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats.

One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs daily; functional observation tests; bodyweight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Accuracy, homogeneity and stability over 4 hours of formulations test substance in propylene glycol were demonstrated by analyses.

Treatment-related findings observed were as follows:

50 mg/kg/day:- Renal hyaline droplets (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (1/5 males).

150 mg/kg/day:- Renal hyaline droplets (all males), increased incidence and severity of tubular basophilia in the kidneys (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (4/5 males).

1000 mg/kg/day:- Slightly lower weight gain on day 8 (males).

-lncreased absolute and relative food intake in weeks 1/2 (males).

-Slightly increased red blood cell count and haematocrit levels (females).

-lncreased liver weights and liver to body weight ratios (females).

-Renal hyaline droplets: (all males), increased incidence and severity of tubular basophilia in the kidneys (3/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (all males).

From the results presented in the report a No Observed Adverse Effect Level (NOAEL) could not be determined for males, but was established to be 150 mg/kg/day for females. However, since the male kidney findings are a species and sex specific response which is not observed in humans, a NOAEL of 150 mg/kg/day may be considered for both sexes.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 July 2003 to 15 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

Deviations:

not specified

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany.
Age at Start of Treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification: Earmark and tattoo.

Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 18.6 - 24.2°C), a relative humidity of 30-70% (actual range: 41 - 88%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type Ill, height 15 cm.) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water: Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Vehicle: Propylene glycol, specific gravity 1.036
Rationale for vehicle: Based on trial formulations performed at NOTOX.
Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance and vehicle.
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purpose of the analytical study was to determine accuracy of preparation, homogeneity and stability of HATCOL 3344 in formulations.
From the results, it was concluded that for the formulations of Groups 1-4 the concentrations analysed were in agreement with target concentrations (91 % to 112 % of target based on peak 1 and 92 % to 113 % of target based on peak 2).
Based on the measurements, formulations of Groups 2 and 4 were considered to be homogeneous.
Formulations of Groups 2 and 4 were found to be stable for 1 hour when stored at room temperature.

Samples
Accuracy, homogeneity and stability after 1, 2 and 4 hours of storage at room temperature were determined for formulations prepared on 15-08-03. Based on the results, accuracy and homogeneity of group 2, accuracy of group 3 and stability of group 2 after 4 hours of storage at room temperature were determined for formulations prepared on 22-09-03.
On 15-08-03 samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks (25, 50 or 100 ml). For determination of accuracy, duplicate samples were taken at 50% height or at 90, 50 and 10% height. For determination of homogeneity, duplicate samples were taken at 90, 50 and 10% height. For determination of stability, duplicate samples were taken at 50% height.
On 22-09-03 samples (approximately 1000 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks (50 or 100 ml). For determination of accuracy, duplicate samples were taken at 50% height or at 90, 50 and 10% height. For determination of homogeneity, duplicate samples were taken at 90, 50 and 10% height. For determination of stability, duplicate samples were taken at 50% height.
The flasks were filled up to the mark with Hexane containing 0.1 mg/l internal standard. The solutions were further diluted with Hexane containing 0.1 mg/l internal standard to obtain concentrations within the calibration range.

Analysis
Analytical method
Several small test substance peaks and three large test substance peaks were observed in the chromatograms of test substance solutions. It was assumed that all peaks derive from the test substance. Quantitative analyses were based on the two largest peaks in the GC-MS chromatogram of HATCOL 3344 (See NOTOX Project 364951 "Development and validation of an analytical method for HATCOL 3344").
Analytical conditions
Column: DB-5-HT, 30 m x 250 µm, dr= 0.1 µm (J&W Scientific, Folsom, CA, USA)
Carrier gas: Helium (constant flow, 1 ml/min)
Injection: TV (Splitless time 2 min; Split flow 25 ml/min; Vent time 0.01 min; Vent flow 50 ml/min)
Injection volume: 200 µl
Injection speed: 2 µl/s
Detection: Mass Spectrometric detection
Ionization source: CI+ (SIM)
MS Interface temperature: 350 °C
Monitored masses: 371 amu for peak 1; 427 amu for peak 2; 437 and 439 amu for the internal standard

PTV program
Initial temperature: 65°C
Initial time: 0.05 min
Rate 1: 720°C/min
Temperature 1: 400°C
Hold time: 15 min

Temperature Program
Initial temperature: 65°C
Initial time: 1 min
Rate 1: 20°C/min
Temperature 1: 360°C
Hold time: 0 min

Calibration
On each day of analysis, calibration solutions in hexane containing 0.1 mg/l internal standard were prepared from two stock solutions in hexane.

Injections
Each individual sample and all calibration solutions were injected in duplicate. All values reported in the tables are the average of the duplicate injections.

Integration
Integration was performed using Chemstation software (Agilent Technologies GmbH, Karlsruhe, Germany) version D.00.00.38, 19-Nov-2001

Calculations
All calculations were performed using non-rounded values. Rounded values are reported in the tables. Therefore, differences may occur when calculating the parameters as mentioned in the tables.

Specifications
Preparation of formulations was considered acceptable if the measured concentration levels are between 90% and 11 0% of the target concentration and if the relative standard deviation is less than 10%. Additionally, formulations are considered to be stable if the relative difference between the t=0 and t=x hour samples is < 10%.

RESULTS
Calibration curves
On each day of analysis, a calibration curve was constructed using seven concentration levels. At each concentration level, two responses were used. The coefficient of correlation was in excess of 0.99 for each calibration curve.

Samples
Maximum deviation between the responses of duplicate injections was less than 10% for each sample unless indicated otherwise.
For all formulations of Group 1 the concentrations analysed were in agreement with the target concentrations.
From the results, it was concluded that for formulation of Group 4 the analysed concentrations were in agreement with target concentrations (93 % to 109 % of target based on peak 1 and 92 % to 105 % of target based on peak 2). For formulation of Group 2 prepared on 15-08-03, the results indicate that there was a homogeneity problem. Therefore, homogeneity and stability of Group 2 was determined again on 22-09-03. For formulation of Group 3 prepared on 15-08-03, the results of duplicate samples were too high (1 19 % and 124 % of target based on peak 1 and 112 % and 1 19 % of target based on peak 2). Therefore, accuracy of Group 3 prepared on 22-09-03 was also determined again on 22-09-03. From the results, it was concluded that for formulations of Group 2 prepared on 22-09-03 the analysed concentrations were in agreement with target concentrations (91 % to 102 % of target based on peak 1 and 94 % to 103 % of target based on peak 2). For formulations of Group 3 the analysed concentrations were in agreement with target concentrations (112 % and 111 % of target based on peak 1 and 113 % and 111 % of target based on peak 2). It was decided to accept these accuracy samples despite the fact that some of the samples were out of the range 90-1 10%.
The formulations of Group 2 and Group 4 were homogeneous (3.7% and 6.7% relative standard deviation based on peak 1 and 3.7% and 5.5% relative standard deviation based on peak 2).
On 15-08-03, stability was determined for Group 2 and Group 4 formulations after 1, 2 and 4 hours of storage at room temperature. This resulted for the 1 hour stability test in a relative difference of 6% and -5% based on peak 1 and 17% and -3% based on peak 2, for the 2 hours stability test in a relative difference of -24% and -2% based on peak 1 and -4% and 1 % based on peak 2 and for the 4 hour stability test in a relative difference of -25% and 11 % based on peak 1 and -2% and 12% based on peak 2. Therefore, the formulations at target concentration of 9.66 and 194 mg/g HATCOL 3344 in propylene glycol were considered stable for 1 hour at room temperature.
On 22-09-03, stability was determined for Group 2 after 4 hours of storage at room temperature.
This resulted in a relative difference of -57% based on peak 1 and -40% based on peak 2. Therefore, the formulations at target concentration of 9.66 mg/g
HATCOL 3344 in propylene glycol were considered not stable for 4 hours at room temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Remarks:

Doses / Concentrations:
0, 50, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per does group

Control animals:

yes, concurrent vehicle

Details on study design:

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Method: Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.
Frequency: Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Positive control:

Postive control not required for this study.

Observations and examinations performed and frequency:

Mortality/Viability: At least twice daily.
Clinical signs: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena (inadvertently, no arena observations were performed on days 22 and 28). The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
Functional Observations: During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Body weights: On days 1,8, 15,22 and 28.
Food consumption: Weekly.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
CLINICAL LABORATORY INVESTIGATIONS: Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post modem examination, between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (1.0 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml). Parameters determined are presented in table form and are attached under Any other information.

Sacrifice and pathology:

NECROPSY
All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution. Parameters determined are presented in table form and are attached under Any other information.

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology, were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
The following slides were examined by a pathologist:
- all tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group;
- all gross lesions of all animals.
All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.

Other examinations:

No further examination specified in the study report.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment.
Incidental findings that were noted included scabs, alopecia, chromodacryorrhoea and broken upper incisors. These findings are more frequently noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted in control males, females dosed at 50 mg/kg/day, animals dosed at 150 mg/kg/day and high dose males.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes were noted in body weights and body weight gain of treated animals.
Minimal weight loss was noted for one control female (no. 24) and one female dosed at 50 mg/kg/day (no. 26) at the end of week 4. A slightly lower mean weight gain was noted for all treated males in weeks 314, while weight gain was also slightly lower for females at 150 mg/kg/day over week 3. These changes occurred in the absence of a dose-response relationship and were therefore considered to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The variation noted in food consumption before or after allowance for body weight between treated and control animals was within the normal range for this type of study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats. White blood cell counts (WBC) of high dose females were reduced. This was considered to be mainly due to a low value for one female (no. 40). The remaining two values for this group were within the normal range for this type of study. Therefore, the deviation was considered to be incidental in nature. Values of other haematological parameters between treated and control animals were similar.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alanine aminotransferase activity values (ALAT) were increased in high dose females (< p0.05).
A high aspartate aminotransferase activity value (ASAT) was noted in one male dosed at 50 mg/kg/day (no. 7). Values of other animals of this dose group were similar to control values. The statistically significant increase of total protein and albumin levels of females at 150 mg/kg/day occurred in the absence of a dose-response relationship. No toxicological relevance was ascribed to these changes.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Description (incidence and severity):

No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with HATCOL 3344, when compared to control animals. Variations noted in motor activity data between treated and control animals occurred in the absence of a dose-related response, similar changes of low or high sensor values, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights and organ:body weight ratios of treated animals were similar to those of control animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy did not reveal any toxicologically relevant alterations. Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, red discolouration of the thymus, mandibular lymph nodes or ovaries, right lateral lobe of the liver grown together with the diaphragm, yellowish-soft nodules on the epididymides, diaphragmatic hernia of the right lateral lobe of the liver, a constricted spleen, red foci on the mandibular lymph nodes, fluid in the uterus, exophthalmus and watery-clear cysts on the ovaries. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Key result

Critical effects observed:

no

FUNCTIONAL OBSERVATIONS SUMMARY

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 1000 MG/KG
MALES
AT WEEK 4
HEARING	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL L	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
STATIC R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
GRIP	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
FEMALES
AT WEEK 4
HEARING	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL L	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
STATIC R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
GRIP	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5

+/++ Steel-test significant 5% (+) or 1% (++) level

 

MACROSCOPIC FINDINGS SUMMARY

	GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 1000 MG/KG
MALES
END OF TREATMENT
Animals examined	5	5	5	5
Animals without findings	1	4	4	3
Animal affected	4	1	1	2
Liver
-Grown together with:	1	0	0	0
Kidneys
-Pelvic dilation	3	0	0	1
Epididymides
-Nodule(s)	0	1	0	0
Thymus
-Discolouration	1	0	1	1
Mandibular l. node
-Discolouration	0	0	0	1
FEMALES
END OF TREATMENT
Animals examined	5	5	5	5
Animals without findings	3	3	2	3
Animals affected	2	2	3	2
Liver
-Diaphragmatic hernia	1	0	1	0
Ovaries
-Cyst(s)	0	0	0	1
-Discolouration	0	0	0	1
Uterus
-Contains fluid	0	0	2	0
Spleen
-Constricted	1	0	0	0
Mandibular l. node
-Focus/foci	0	1	0	0
-Discolouration	1	1	1	1
Eyes
-Right side exophthalmus	0	0	0	1

#/## Fisher’s Exact test significant at 5% (#) or 1% (##) level

Conclusions:

Based on the results presented in the report, a NOAEL of 1000 mg/kg/day for HATCOL 3344 was established.

Executive summary:

Subacute 28-day oral toxicity with HATCOL 3344 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

The study was based on the following guidelines: EC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996. OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995-OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats.

One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Accuracy and homogeneity of formulations of test substance in propylene glycol were acceptable. Group 2 formulations (50 mg/kg/day) were not stable over a 4-hour period (approximately 50% recovery), while stability of group 4 formulations (1000 mg/kg/day) was confirmed over the same time-period. Based on overall results and the established NOAEL, it was considered that sufficient analytical support was obtained for the purpose of this study.

Treatment-related findings observed were as follows:

50 mg/kg/day: No treatment-related findings noted.

150 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: No treatment-related findings noted.

CONCLUSION

Based on the results presented in this report, a NOAEL of 1000 mg/kg/day for HATCOL 3344 was established.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07 July 2003 to 04 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU and US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (71 Ol), EPA 712-C-00-366, 2000.

Deviations:

not specified

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EC). Source: Charles River Deutschland, Sulzfeld, Germany.
Age at Start of Treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification: Earmark and tattoo.

Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 ± 3°C (actual range: 18.6 - 23.7°C) a relative humidity of 30 - 70% (actual range: 41 - 81 %) and 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal level of 70% for relative humidity. Based on laboratory historical data these conditions were considered not to have affected the study integrity.
Accommodation: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type Ill, height 15 cm.) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water: Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Vehicle: Propylene glycol, specific gravity 1.036
Rationale for vehicle: Based on trial formulations performed at NOTOX.
Stability of test substance in vehicle: At least 1 hour (determined at NOTOX).
Method of formulation: Initially, formulations (w/w) were prepared daily within 4 hours prior to dosing. However, results from stability analyses showed that formulations were stable for at least 1 hour at room temperature. Therefore, from 21 July (beginning of week 3) onwards formulations were prepared within approximately 1.25 hours prior to dosing. Formulations were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 2 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 4 hours was also determined (highest and lowest concentration). After the in-life phase additional analyses were performed to check accuracy of preparation (all dose groups) and stability over 1 and 2 hours (highest and lowest concentration). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX Project 364949).

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily.

Remarks:

Doses / Concentrations:
0, 50, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per dose group (20 males and 20 females in total).

Control animals:

yes, concurrent vehicle

Details on study design:

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Method: Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.
Frequency: Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Positive control:

Postive control not required in this study.

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.
Clinical signs: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
Functional Observations: During week 4 of treatment, motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England) was performed on all animals.
Body weights: On days 1,8, 15,22 and 28.
Food consumption: Weekly.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

CLINICAL LABORATORY INVESTIGATIONS: Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled postmortem examination, between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (1.0 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml).Parameters determined reported in table form – attached under Any other information.

Sacrifice and pathology:

NECROPSY: All animals were deeply anaesthetised the end of the observation period using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:
Identification marks: not processed, Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions.

ORGAN WEIGHTS: The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY: The following slides were examined by a pathologist:
- all tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group;
- all gross lesions of all animals.
All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.

Other examinations:

No further examinations detailed in the study report.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one-t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. - The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment.
Greasy coat and red discolouration of the fur was noted in two females treated at 1000 mg/kg/day on two days during the third treatment week. Since these findings were only incidental, these effects were considered not to be treatment related. Other incidental findings that were noted included alopecia and watery discharge from the eye. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

Mortality:

no mortality observed

Description (incidence):

Mortality: No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Red blood cell count (RBC) was increased in females at 1000 mg/kg/day.
The decrease of partial thromboplastin time (APTT) in males at 150 mg/kg/day and the decrease of prothrombine time (PT) in females at 150 and 1000 mg/kg/day was considered not to be toxicologically relevant considering the direction of the change (i.e. a decrease) and/or the lack of an effect at the highest dose level.
In females, the lower white blood cell count and higher neutrophil count at 50 and 150 mg/kg/day occurred in the absence of a dose-response relationship and therefore considered not to be related to treatment with HATCOL 5236.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholesterol values were reduced in males at 1000 mg/kg/day.
Changes in creatinine, total protein values, calcium concentration and albumin concentration in males at 50 and 150 mg/kg/day, respectively were considered to be of no toxicological significance in the absence of a treatment-related distribution.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights and organ to body weight ratios of treated animals were considered to be similar to those of control animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlargement of the liver was noted in two males at 1000 mg/kg/day.
Incidental findings among control and treated animals included an accentuated lobular pattern of the liver, pelvic dilation of the kidneys, reduced size of testes and epididymides, reddish or red foci on thymus, red discolouration of the thymus and of the mandibular lymph nodes, caecum and colon distended with gas, adrenals grown together with kidneys and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Other effects:

not specified

Details on results:

Analysis of Dose Preparations: Analyses of HATCOL 5236 in propylene glycol were based on two peaks observed in the GCMS chromatogram. Test substance formulations formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 88% and 119% of target. Although a few values were slightly outside the nominal range of 100 ± 10%, mean values were within this range.
Results from additional analyses of formulations revealed accuracy values between 103% and 120%. No explanation was found for these slightly higher values. Based on the overall accuracy results, it was concluded that an acceptable level of accuracy for this study was achieved.
Group 4 formulations were stable for at least 2 hours at room temperature, and analyses of these formulations after 4 hours storage at room temperature showed results slightly below the limit of 90% (i.e. 89%). Stability analyses of group 2 formulations showed that the formulations were stable for at least 1 hour at room temperature. However, stability measurements of these formulations showed a recovery of 63% and 71 % after a 2-hour recovery period and a recovery of 58% and 65% after a 4-hour period. Based on these results it was decided to reduce the time between formulating and dosing to a maximum of 1.25 hours, starting at the beginning of week 3. On a few occasions during the study time between preparation of the formulations and dosing (slightly) exceeded 1.25 hours.
The No Observed Adverse Effect Level (NOAEL) in this study was established at 1000 mg/kg/day and stability of formulations of 1000 mg/kg/day over 4 hours was only slightly below the acceptable limit. Therefore, it was concluded that sufficient evidence that animals were dosed at least very close to the target dose of 1000 mg/kg was available and that sufficient analytical support was obtained for the purpose of this study.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; water consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

no

Conclusions:

No Observed Adverse Effect Level (NOAEL) for HATCOL 5236 of 150 mg/k/day was established. Based on the absence of functional or morphological disturbances supporting higher red blood cell count and lower cholesterol values in the high dose females and males respectively, a NOAEL of 1000 mg/kg/day may be considered.

Executive summary:

Subacute 28-day oral toxicity with HATCOL 5236 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

 

The study was based on the following guidelines:EC Directive 96154/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996. OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995. OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

 

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats.

One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

 

RESULTS

Accuracy, homogeneity and stability over at least 1 hour (50 mg/kg formulations) or at least 2 hours (1000 mg/kg formulations) of formulations of test substance in propylene glycol were demonstrated by analyses.

50 mg/kg/day: No treatment-related findings noted.

150 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: Higher red blood cell count (F), lower cholesterol values(M).

 

CONCLUSION

The higher red blood cell count and the lower cholesterol values found in the high dose females and males respectively, were not supported by other changes in blood parameters or histopathological lesions. Therefore, the toxicological relevance of these effects is doubtful. An enlarged of the liver was noted in two males at 1000 mg/kg/day. Since this was not seen in the other high dose animals and since no morphological correlates were found, this finding was considered not to be toxicologically relevant.

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for HATCOL 5236 of 1 50 mg/kg/day was established. Based on the absence of functional or morphological disturbances supporting higher red blood cell count and lower cholesterol values in the high dose females and males respectively, a NOAEL of 1000 mg/kg/day may be considered.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(adopted 1998)

Deviations:

yes

Remarks:

no analytical purity reported; the low and intermediate dose differ from each other for a 20 fold interval

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Details on strain: Crl:CD (SD) BR- Source: Charles River (UK) Limited, Margate, Kent- Age at study initiation: approx. 6-8 weeks old- Weight at study initiation: males: 201 - 276 g; females: 148 - 203 g- Housing: in groups of up to four by sex in polypropylene grid-floor cages- Diet: pelled diet (Rodent 5LF2 (Certified) Diet, International Product Supplies Ltd., Northants, UK), ad libitum- Water: drinking water, ad libitum- Acclimation period: 13 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 21 ± 2- Humidity (%): 55 ± 15- Air changes (per hr): at least 15- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

dried, BP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:- Formulations were prepared weekly and stored at aapprox. +4°C in the dark.VEHICLE- Name: dried Arachis oil BP- Concentration in vehicle: 1.25 mg/mL (low dose), 12.5 mg/mL (intermediate dose), and 250 mg/mL (high dose) - Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. The results show that the formulations were stable for at least fourteen days.Samples were taken of each test material formulation and were analysed for concentrations of the test substance by high performance liquid chromatography (HPLC) using an external standard technique (UV detector). The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:5, 50, and 1000 mg/kg bw/dayBasis:actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a range-finder study. 1 group of 6 rats (3 males and 3 female) was administered by gavage with 1000 mg/kg bw test substance, while control animals (3 males and 3 female) were treated in an identical manner with dried arachis oil BP. The oral route was selected as the most appropriate route of exposure, based on the physical prperties of the test material.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: immediately before dosing, and one hour after dosing; additionally on working days 5 hours after dosing- Parameters: overt signs of toxicity, ill-health or behavioural changeDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity.- Parameters: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, piloerection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.BODY WEIGHT: Yes - Time schedule for examinations: on Day 0, at weekly intervals thereafter, and at terminal sacrifice.FOOD CONSUMPTION:- Food consumption for each animal determined: Yes- Food consumption for each cage group: at weekly intervalsWATER CONSUMPTION: Yes - Time schedule for examinations: daily for each cage group (by visual inspection of the water bottles)OPHTHALMOSCOPIC EXAMINATION: Yes - Time schedule for examinations: pre-treatment and before termination of treatment (during week 12)- Dose groups that were examined: all control and high dose animals- Parameters: observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 1.0% "Mydriacyl" solution, a detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.HAEMATOLOGY: Yes - Time schedule for collection of blood: on Day 90- Anaesthetic used for blood collection: No- Animals fasted: No - How many animals: all surviving animals- Parameters: haemoglobin (Hb), erythrocyte count (RBC), haematocrit (Hct), erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), total leucocyte count (WBC), differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), platelet count (PLT), reticulocyte count (Retic) (cresyl blue stained slides prepared, but not assessed), prothrombin time (CT), activated partial thromboplastin time (APTT).CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: on Day 90- Animals fasted: No - How many animals: all surviving animals- Parameters: urea, glucose, total protein (Tot.Prot), albumin, albumin/globulin (A/G) ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili).URINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: during week 12 functional performances tests were performed on all surviving animals, together with an assessment of sensory reactivity to different stimuli.- Dose groups that were examined: all- Battery of functions tested: sensory activity / grip strength / motor activity - Motor activity was assessed by 20 purpose built 44 infra-red beam automated activity monitors. Evaluation period was 16 hours for each animal.- Grip strength (forelimb, hindlimb) was determined by means of an automated grip strength meter. Three consecutive trials were performed for each animal.- Sensory activity to auditory, visual and proprioceptive stimuli. Parameters: grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex, blink reflex.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes On completion of the dosing period all surviving animals were sacrificed and subjected to a full external and internal examination.The following organs were dissected free from fat and weighed before fixation: adrenals, brain, epididymes, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus.HISTOPATHOLOGY: Yes Samples from the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, ileum (including Peyer's patches), jejunum, rectum, epididymides,eyes, gross lesions, heart, kidneys, liver, lungs (with bronchi), trachea, lymph nodes (cervical and mesenteric), mammary glands, ovaries, uterus, muscle (skeletal), pancreas, pituitary, prostate, seminal vesicles, sciatic nerve, spinal cord (cervical, mid-thoracic and lumbar), skin (hind limb), spleen, oesophagus, salivary glands (submaxillary), stomach, testes, thymus, thyroid, urinary bladder.

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney'U' test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased salivation was detected immediately after dosing for 1000 mg/kg bw/day females from Day 17 and for 1000 mg/kg bw/day males from Day 18 onwards. Hunched posture was observed for individual 1000 mg/kg bw/day females from Day 37 with isolated incidents of tiptoe gait also observed on Days 37 and 38 and exophthalmia present in two females from Day 75 and 76 onwards. Exophthalmia was also evident in two 50 mg/kg bw/day females from Day 81 and hunched posture was observed for another 50 mg/kg bw/day female from Day 81 onwards. No such observations were detected for animals of either sex treated with 5 mg/kg bw/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related deaths during the 90-day study. One male treated with 5 mg/kg bw/day was found dead on Day 36. There was no evidence to indicate this death was attributable to test material toxicity, the most plausible cause of death was a likely association with gavage mis-administration. One male treated with 50 mg/kg bw/day was terminated on Day 53 following observations indicative of a mal-dose. Neither death was considered to be treatment-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically adverse effect on body weight development was detected during the study. Females from all treatment groups showed low weight gains compared with controls during Week 9. However in isolation and in the absence of a convincing dose dependent trend, this was considered not to be toxicologically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no toxicologically important adverse effect on food consumption during the study. However, a slight reduction in food consumption was observed for all female test groups at Week 9 returning to similar levels to controls thereafter in consideration of which these effects were considered not to be of toxicological relevance.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no toxicologically important adverse effect on food efficiency during the study. However, a slight reduction in food efficiency was observed for all female test groups at Week 9 returning to similar levels to controls thereafter in consideration of which these effects were considered not to be of toxicological relevance

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related ocular effects were detected.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes detected in the haematological parameters measured. Females from all treatment groups showed statistically significant reductions in leucocyte count, specifically in the lymphocyte fraction, but these were considered attributable to higher than expected control values and not a consequence of treatment. Furthermore, 50 mg/kg bw/day males showed a reduction in haemoglobin concentration, however, in the absence of a convincing dose response and minimal statistical significance achieved (p<0.05), this value was considered to have arisen fortuitously. The reduction in erythrocyte count detected for 1000 mg/kg bw/day males achieved a statistical significance of p<0.01, however, in isolation, it was considered unrelated to treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected for the blood chemical parameters investigated. Statistical analysis did not reveal any statistically significant intergroup differences.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioural assessment: Weekly open field arena observations confirmed the clinical signs of hunched posture, and exophthalmia detected for females treated with 1000 mg/kg bw/day from week 6 and for 50 mg/kg bw/day females from Week 12 onwards. Exophthalmia was also observed for 1000 and 50 mg/kg bw/day females from week 12 during the study and an incident of noisy respiration was seen for one 50 mg/kg bw/day female during the Week 12 assessment. No such effects were detected for males of the high and intermediate dose groups and for animals of either sex treated with 5 mg/kg bw/day.
Functional Performance Tests: No treatment-related changes were detected.
Sensory Reactivity Assessments: No treatment-related changes were detected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were detected. Liver weight, relative to terminal bodyweight was elevated of about 13% for animals of either sex treated with 1000 mg/kg bw/day (p<0.01). The toxicological significance of this finding is doubtful because of the absence of supporting biochemical or histopathological changes and therefore suggests that the organ weight increase is not a direct consequence of treatment. No such observations were detected for animals of either sex treated with 50 or 5 mg/kg bw/day

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected. The decedent 5 mg/kg bw/day male showed dark kidneys and dark foci present on the liver and lungs and pallor of the gastric epithelium. These observations are characteristic of autolytic post mortem changes. The male terminated on Day 53 showed a mass of food present on the right side of the sternum and a liver pallor. In the absence of histopathological data to suggest an effect of treatment, this was considered attributable to the administration procedure and not a toxic effect of the test material.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in any of the tissues examined. There were no histopathological indications as to the cause of death for the 5 mg/kg bw/day male found dead during the study, but histopathological findings for the 50 mg/kg bw/day male suggested the animal may have suffered internal trauma as a consequence of mal-dosing. In general, the contribution made by interim death animals to a group assessment of treatment-related pathology should be regarded with caution, since animals dying before termination of the dosing period would not have received their full exposure to the test material. Both of these animals were excluded from statistical analyses. A variety of spontaneous histopathological changes were encountered, some of which warrant specific comment as follows:
HEART: Focal myocarditis was observed in several control and treated animals and is a common background entity in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.
LIVER: Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.
SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.
KIDNEYS: Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance as the severities or frequencies reported in this study. Similarly focal corticomedullary mineralization is a commonly observed background condition amongst female rats.
OESOPHAGUS: Inflammatory infiltration of the peripheral oesophageal musculature was observed in a few animals. This is a relatively frequently observed consequence of trauma of gavage dosing.
LUNGS: A minimal, and rarely slight, severity of bronchus associated lymphoid tissue was reported for most animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis are also a commonly observed pulmonary change in laboratory maintained rats of this age and are not suggestive of significant respiratory disease. A rather greater prevalence than might normally be expected of alveolar macrophage accumulations was seen among male rats, particularly control animals.
PROSTATE: Interstitial chronic inflammatory cell infiltrates are a commonly observed background finding in laboratory maintained rats.
BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.
UTERUS: Dilatation of the uterine horns is a commonly observed cyclical condition in laboratory maintained female rats.
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence of severity between control and treatment groups, all were considered to be without toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL corresponds to the highest dose level tested.

Critical effects observed:

not specified

Conclusions:

NOAEL 1000 mg/kg bw/day (actual dose received) is assigned. The NOAEL corresponds to the highest dose level tested. No significant effects were observed.

Executive summary:

NOAEL 1000 mg/kg bw/day (actual dose received) is assigned. The NOAEL corresponds to the highest dose level tested. No significant effects were observed.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Nov - Dec 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Zeneca Pharmaceuticals, Alderly Park, Macclesfield, Cheshire, UK- Age at study initiation: 28 d- Weight at study initiation: 148.45 g (males); 122.6 g (females)- Housing: sexes separately, five per cage, cages had measurements of 26.5 x 50.0 x 20.0 cm and were constructed of stainless steel mesh with one solid side.- Diet: CT1 diet; Special Diets Services Limited, Witham, Essex, UK- Water: tap water, ad libitum- Acclimation period: approx. 1 weekENVIRONMENTAL CONDITIONS- Temperature (°C): 19-23- Humidity (%): 45-65 (71 at one occasion)- Air changes (per hr): 25-30- Photoperiod (hrs dark / hrs light): 12 / 12IN-LIFE DATES: From: November 1992 To: December 1992

Route of administration:

oral: feed

Vehicle:

other: in diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: All diet preparations were based on CT1 diet (Special Diets Services Limited, Witham, Essex, UK). They were prepared by grinding the appropriate amount of test substance with 1 kg of milled CT1 diet. This premix was then added to 14 kg of diet and mixed thoroughly with a Pharma Blender Model PMA 100S (T K Filder).DIET PREPARATION- Rate of preparation of diet (frequency): 15 kg batches- Mixing appropriate amounts with (Type of food): CT1 diet (Special Diets Services Limited, Witham, Essex, UK)- Storage temperature of food: - 20°C, stored at room temperature for usage up to 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical stability was determined for diet preparations over a period of 5 weeks following storage at room temperatureT or at -20°C.Samples were extracted by chemical shaking with ethyl acetate. The supernatant was diluted with ethyl acetate to give solutions containing appropriate concentrations of the test substance. Extracts were analysed by gas chromatography using flame ionisation detection. The extract concentration was calculated by reference to data from a standard containing a known concentration.

Duration of treatment / exposure:

daily

Frequency of treatment:

28 d

Remarks:

Doses / Concentrations:1000 ppm, 5000 ppm, 12500 ppmBasis:nominal in diet

Remarks:

Doses / Concentrations:112, 562, 1450 mg/kg bw/dBasis:other: actual ingested for males

Remarks:

Doses / Concentrations:119, 586, 1613 mg/kg bw/dBasis:other: actual ingested for females

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on results of preliminary feeding studies

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: Daily- Cage side observations checked: changes in clinical condition and behaviour and significant changes were recorded.DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: on Days 8, 15, 22, 29- observations included, but were not limited to the assessment of autonomic function (e.g. lacrimation, salivation, piloerection, exophthalmus, urination, defecation, pupillary function, ptosis); description, incidence and severity of any convulsions, tremors, abnormal motor function, alteration in respiration, reactivity to stimuli, changes in the level of arousal, sensorimotor responsesBODY WEIGHT: Yes, measurement in replicate order immediately before feeding and at the same day once a week until termination.FOOD CONSUMPTION AND COMPOUND INTAKE: - Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight: Yes, on a weekly basis- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: YesHAEMATOLOGY: Yes At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Hemoglobin, red cell count, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, platelet count, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, prothrombin time and kaolin-cephalin timeCLINICAL CHEMISTRY: Yes At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Albumin, total protein, cholesterol, triglycerides, urea, creatinine, glucose, total bilirubin and alkaline phosphatase, plasma gamma-glutamyl transferase, plasma alanine aminotransferase, plasma aspartate aminotransferase, plasma creatine kinase, plasma sodium, plasma potassium, plasma chloride, plasma calcium and plasma phosphorusNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: on Days 8, 15, 22, 29- Dose groups that were examined: All- Battery of functions tested: sensory activity / grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: YesHISTOPATHOLOGY: Yes (adrenals, aorta, bladder, bone and bone marrow (femur), brain, caecum, colon, cervical lymph node, cervix, colon, duodenum, epididymis, eye and harderian gland, heart, ileum, jejunum, kidney, liver, lungs, mammary gland, mesenteric lymph node, nasal passages, oesophagus, oral cavity, ovaries, pancreas, parathyroid glad, pituary gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skin, spinal chord, spleen, sternum, stomach, testes, thymus, thyroid gland, trachea, uterus, voluntary muscle)

Statistics:

Bodyweights were considered by analysis of covariance on initial body weight, separately for males and females. Time to tail flick and fore and hindlimb grip strength at weeks 2, 3, 4 and 5 were considered by analysis of variance, separately for both sexes.Haematological and clinical blood parameters were considered by analysis of variance.Organ weights were considered by analysis of variance and covariance on final body weight separately for both sexes.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

There were no mortalities.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant effects on body weight and all final bodyweights were within 3% of the respective controls, after adjusting for initial weight differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in all treated groups remained similar to, or exceeded that, of the respective control group throughout the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were statistically significant reductions in haemoglobin and haematocrit at 12.500 ppm in male rats. Statistically significant reductions in haemoglobin and haematocrit were seen in females at 1000 and 5000 ppm and in white blood cell count at 1000 ppm. In the absence of a coherent dose-response relationship, these differences were considered incidental to treatment. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were minor reductions in plasma cholesterol, triglyceride and total protein levels and plasma alanine transferase activities in males at 12500 ppm compared to controls. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

A slightly reduced splay reflex was observed in one female of the 1000 ppm group (on days 29 and 30), in one male of the 5000 ppm group (on day 29) and in one male of the 12.500 ppm group (on day 29). As isolated observations, these were considered to be incidental. There were no differences in time to tail flick in either sex which could be attributed to treatment. The statistically significant increase in time to response observed on day 22 for males (5000 ppm) and day 8 for females (1000 ppm) were considered to be incidental to treatment in the absence of similar changes at higher dose levels. There was no evidence of any treatment related effects on forelimb or hindlimb grip strength. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights adjusted for body weight were statistically significant increased in males at 5000 and 12500 ppm. All the females in the treatment groups had slightly raised kidney weights compared to control, but none achieved statistical significance, and there was no evidence of a coherent dose response relationship. Liver weights adjusted for body weight were statistically significant increased in both sexes at 12500 ppm and in males at 5000 ppm. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings were apparent at the end of the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related findings were present in the kidney of male rats from all dose groups. In the 5000 and 12500 ppm dose group these comprised increased tubular hyaline droplet formation and tubular basophilia in all animals, and granular cast formation in four of the 5000 ppm animals and all of the 12500 ppm animals; the latter occurring at the cortico-medullary injection. In the 1000 ppm group, increased renal hyaline droplet formation and/or tubular basophilia were seen, but not granular cast formation. In the liver, there was minimal hepatocyte hypertrophy in 4/5 male rats in the 12500 ppm group. The increased kidney weights and microscopic findings of renal tubular basophilia, granular cast formation and increased hyaline droplet formation present in male rats at 5000 and 12.500 ppm are clearly treatment related. These findings are consistent with the well characterized light hydrocarbon nephropathy described for male rats, following to a variety of chemicals including light hydrocarbons such as unleaded gasoline and trimethyl pentane. The characteristics include an increased accumulation of hyaline droplets in male rat kidneys, the main constituent of which is alpha 2µ-globulin (Alden et al. Adv. Modern Environ Toxicol 7: 107-120 (1984); Stonard et al. Renal Heterogeneity and Target Cell Toxicity. Bach PH and Lock EA Eds, John Wiley and Sons (1985)). It is widely accepted that this phenomenon is specific to male rat and as such appears to have no relevance for man (Swenberg et al. Toxicol and App. Pharmacol. 97: 35-46 (1989)).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

DIET ANALYSIS: All diets prepared were found to be within 4% of the target concentration. The homogeneity of the test material in the diet, determined at 1000 and 12500 ppm inclusion levels was within 2 % of the overall mean concentration for both levels. Chemical stability of the test material, assessed at the 1000 and 12500 ppm inclusion levels stored at room temperature or at -20 °C was satisfactory over the period of use. Dose rates (based on nominal dietary levels) were highest at the start of the study and declined rapidly during the period of rapid growth to week 4.

Key result

Dose descriptor:

NOAEL

Effect level:

1 613 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed in female rats

Key result

Dose descriptor:

NOAEL

Effect level:

1 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to 12500 ppm

Critical effects observed:

not specified

This study is read across from closely related structural analogue, CAS 68424-31-7. The structure of the read-across substance is provided under 'illustration' below.

Reference 6

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Nov 1997 - 16 Mar 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

ToxLabs Prüflabor GmbH, Greppin, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 32-38 d
- Weight at study initiation: 148.5 g (mean value males), 136.7 g (mean value females)
- Housing: one or two animals in cages (Makrolon Type 3)
- Diet: Altromin 1326, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 30-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: distilled water containing 1% Tween 80

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 1%
- Lot/batch no. (if required): S23350 739

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 2 mL samples of each formulation were taken and stored in the frozen state until measurement

Duration of treatment / exposure:

90 d

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 (control, test and satellite groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selecting satellite groups: 10 animals each from the high dose and the vehicle group were used to investigate reversibility of possible effects
- Post-exposure recovery period in satellite groups: 28 d

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, autonomic activity, presence of clonic or tonic movements, stereotypies, bizarre behavior
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes, changes in skin, fur, eyes, mucous membranes, gait, posture: response to handling; occurrence of secretions and excretions
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly from the start to the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the administration and at the end of the study
- Dose groups that were examined: All (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study
- Anesthetic used for blood collection: Yes (Ether)
- Animals fasted: Yes, over night
- How many animals: All animals
- Parameters examined: erythrocyte count, hemoglobin concentration, packed cell volume, platelet count, total leukocyte count, leukocyte differential count, prothrombine time, fibrinogen concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study (including the satellite groups)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: alkaline aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, creatinine, fasting glucose, phosphorus, total cholesterol, total protein, albumin, chloride, potassium, sodium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to administration, at monthly intervals and in the last week of dosing and in the last week of the recovery period.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (auditory, visual and proprioceptive stimuli) / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: cranial, thoracic and abdominal cavities were opened and examined macroscopically
HISTOPATHOLOGY: adrenals, aorta, brain (3 sections), epididymides, eye, femur, heart, kidney, liver, lungs (incl. mainstem bronchi), mesenteric lymph node, muscle incl. sciatic nerve, oesophagus, ovaries, pancreas, pituitary, prostate, seminal vesicle, skin incl. mammary glands, small and large intestine (including peyer´s patches), spinal chord (3 levels), spleen, sternum with bone marrow, stomach, submandibular lymph node, testes, thymus, thyroid (incl. parathyroids), trachea, urinary bladder, uterus

Other examinations:

Organ weights of adrenals, brain, epididymides, testes, heart, kidneys, liver, ovaries, spleen, testes and thymus

Statistics:

Body weights, food consumption: Welch t-test

Haematology, coagulation, clinical biochemistry and absolute and relative organ weights: Dunnett´s test

Differential leukocyte count: Mean, range and standard deviation

Clinical signs:

no effects observed

Description (incidence and severity):

None of the animals showed any alterations of their general state of well-being and behaviour at any observation period (few observations were made substance independent and for a short period of time).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two animals died shortly after administration due to incorrect gavage shown by lungs filled with blood.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Not affected by the test compound.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Not dose-dependently influenced

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No alterations.

Haematological findings:

no effects observed

Description (incidence and severity):

Not influenced.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The fibrinogen concentration of the plasma was increased in the female animals of the high dose group, this was no longer apparent at the end of the treatment-free period.
The activity of alkaline phosphatase of the serum significantly increased in the high dose group, males and femals. This indicates damage to liver cells and/or an increased function rate. This finding was no longer apparent at the end of the treatment-free period.
The serum urea nitrogen was significantly increased in the middle and high dose group of the males and in the high dose group of female animals. The creatinine content was significantly increased in all male and in the high dose group of the female animals. The phosphorus content was significantly increased dose-dependently in all female animals and the sodium content was dose-dependently decreased in the male animals, significantly in the animals of the middle and high dose groups. These effects were no longer apparent at the end of the treatment-free period.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes in grip strength, motor activity and sensory response.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative kidney weights were increased in all male animals in the high dose group which was still present after the recovery period. Absolute and relative liver weights were increased in both sexes but this was no longer apparent after the recovery period in females. Other significant differences seem to be incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No substance-dependent changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Intracellular lipid droplets in hepatocytes of the female animals in the high and mid dose group (5-90% of the observed area) with cell lesions were clearly caused dose-dependently by the test article. There was no special localization of the changes of hepatocytes in the liver lobules. In most cases only low grade intracellular lipopexia occurred in the male animals.
Stomach: Oesophagal part and cardia with multilayered squamous epithelium, leukocyte infiltration in the submucosa and fibrous repair, fibrotic regions in the submucosa of the glandular stomach
Lungs: Atelectactic and emphysemic areas
Thymus: Partial substitution of the parenchyma by fibrinogenesis
Skin: Benign fibrous proliferation
Sciatic nerve: Thickening of the perineurium and thickening of the adventitia of the vessels

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Key result

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (limited parameters examined, no daily observation, no data on test substance purity)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

(limited parameters examined, no daily observation)

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Taconic, Germantown, NY, USA- Weight at study initiation: males: 379-388 g ; females: 234-239 g- Housing: animals were housed in the exposure chambers (feed and water was removed during exposure)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: 1.0 µm/ approx. 1.8

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: 1000 L stainless steel and glass exposure chambers; chambers contained catch pans between each of three leveles of cages. - System of generating particulates/aerosols: The test material was aerosolized directly from the liquid by a modified Lakin nebulizer on each chamber. The test material was in a straight-walled glass flask and the barrels of the nebulizer were immersed under the level of the liquid in order to maximize the amount of material generated. The distance from the nebulizer to the walls of the flask was approx. 3 cm. After exiting the flask, the aerosol passed through a glass impactor to remove most of the larger particles. The remaining aerosol was mixed with the main air stream for each chamber before entering the chamber. - Temperature and humidity in air chamber (by a Taylor wet/dry bulb hydrometer approx. every 30 min during each exposure): approx. 23 °C, 56 - 64% - Air flow rate: approx. 300 L/min (mean chamber flow per group: 297, 308, 342, and 243 L/min, respectively)TEST ATMOSPHERE- Brief description of analytical method used: gravimetric sampling on glass fiber filters (3 times during each exposure); some filters were additionally analyzed by GC/MS- Samples taken from breathing zone: yesNominal concentrations were determined as the loss of weight of fluid from the generator divided by total air flow through the chamber.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day5 days/week

Remarks:

Doses / Concentrations:0.00 ± 0.00, 0.05 ± 0.01, 0.17 ± 0.01, and 0.56 ± 0.02 mg/LBasis:analytical conc.

Remarks:

Doses / Concentrations:0.05, 0.15, and 0.5 mg/LBasis:nominal conc.

No. of animals per sex per dose:

15(Additional 10 male rats per group were included for examination of pulmonary function tests and analysis of pulmonary hydroxyproline following exposure.)

Control animals:

yes, concurrent no treatment

yes, sham-exposed

Details on study design:

- Dose selection rationale: The highest dose was expected to result in abnormal accumulation of test material in the lung and possible impairment of normal clearance mechanisms. The low dose is a factor of 10 above the TLV (treshold limit value) for mineral oil mistes, no untoward effects were expected at this level.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: daily (except weekends)DETAILED CLINICAL OBSERVATIONS: No BODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: NoFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data:No WATER CONSUMPTION: No OPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: Yes- How many animals: all core animals- Parameters examined: complete blood count (CBC) (white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and platelets) and differential count.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: Yes- How many animals: all core animals - Parameters examined: glucose, urea nitrogen, total protein, albumin (A), globulin (G), A/G, sorbitol dehydrogenase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total bilirubin, creatinine colesterol, triglycerides, uric acid, Cl, Ca, Na, K, and PURINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: NoOTHER:Lung function: The animals were anaesthetized and pulmonray function tests were performed (deflation quasistatic pressure-volume cureved, functional residual capactiy, and maximal forced exhalation maneuver). After the pulmonray function tests, the lungs were removed and all lobes were weighed. Lobes were frozen for analysis of hydroxyproline content and analysis of test material remaining in the lung.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; organ weights (adrenals, kidney, spleen, brain, liver, testes, epididymides, ovaries, thymus, heart, prostate, uterus, right middle lung lobeHISTOPATHOLOGY: Yes (untreated and high-dose): adrenals, ovaries, sternum, pancreas, brain, salivary gland, eye, spleen heart, stomach, colon, testes, duodenum, thymus, kidneys, thyroid, liver, tracheobronchial lymph nodes, lung, nasal turbinates, thigh muscle, urinary bladder, sciatic nerve, and any gross lesions. Only the lungs and tracheobronchial lymph nodes of the untreated controls were processed. 10 males of group 1, 2 and 5 (untreated, sham-exposed, and high-dose) were evaluated for morphology, number of sperm and number of testicular spermatids.

Statistics:

ANOVA and Tukey´s multiple range test: body weighs, male reproductive endpoints, haematology, and serum chemistryANOVA and Duncan´s multiple range test: organ weights, pulmonary function, and pulmonary hydroxyproline

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed.

Mortality:

no mortality observed

Description (incidence):

No mortalities observed.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Increased body weights were observed in treated males. The difference compared to control was statistically significant, but as no clear dose-response was seen and the difference was lower than 7%, it was not considered to be of toxicological relevance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The lungs had a minimal increase in weight after exposure to 0.50 mg/L. Other organ weights were not affected by exposure to the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The number of macrophages in the pulmonary alveoli increased slightly. This increase was small considering the high (500 mg/nr) aerosol concentration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of the lungs of animals in the high-dose group revealed one to two plump macrophages with sparse cytoplasmic vacuoles in less than 1.0% of the aveoli (in controls less than 0.1% would be expected). OTHER FINDINGS- Sperm morphology: No treatment-related effects were noted in sperm morophology or in sperm and spermatid counts. - Lung function: There were no significant differences between any groups for any of the pulmonary function parameters. The only parameter affected by exposure was the total weight of the five lung lobes. Weight for the high-dose group was significantly greater than the other groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEC

Effect level:

0.5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

0.5 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (limited parameters examined, no daily observation, no data on test substance purity)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

(limited parameters examined, no daily observation)

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Taconic, Germantown, NY, USA- Weight at study initiation: males: 379-388 g ; females: 234-239 g- Housing: animals were housed in the exposure chambers (feed and water was removed during exposure)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: 1.0 µm/ approx. 1.8

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: 1000 L stainless steel and glass exposure chambers; chambers contained catch pans between each of three leveles of cages. - System of generating particulates/aerosols: The test material was aerosolized directly from the liquid by a modified Lakin nebulizer on each chamber. The test material was in a straight-walled glass flask and the barrels of the nebulizer were immersed under the level of the liquid in order to maximize the amount of material generated. The distance from the nebulizer to the walls of the flask was approx. 3 cm. After exiting the flask, the aerosol passed through a glass impactor to remove most of the larger particles. The remaining aerosol was mixed with the main air stream for each chamber before entering the chamber. - Temperature and humidity in air chamber (by a Taylor wet/dry bulb hydrometer approx. every 30 min during each exposure): approx. 23 °C, 56 - 64% - Air flow rate: approx. 300 L/min (mean chamber flow per group: 297, 308, 342, and 243 L/min, respectively)TEST ATMOSPHERE- Brief description of analytical method used: gravimetric sampling on glass fiber filters (3 times during each exposure); some filters were additionally analyzed by GC/MS- Samples taken from breathing zone: yesNominal concentrations were determined as the loss of weight of fluid from the generator divided by total air flow through the chamber.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day5 days/week

Remarks:

Doses / Concentrations:0.00 ± 0.00, 0.05 ± 0.01, 0.17 ± 0.01, and 0.56 ± 0.02 mg/LBasis:analytical conc.

Remarks:

Doses / Concentrations:0.05, 0.15, and 0.5 mg/LBasis:nominal conc.

No. of animals per sex per dose:

15(Additional 10 male rats per group were included for examination of pulmonary function tests and analysis of pulmonary hydroxyproline following exposure.)

Control animals:

yes, concurrent no treatment

yes, sham-exposed

Details on study design:

- Dose selection rationale: The highest dose was expected to result in abnormal accumulation of test material in the lung and possible impairment of normal clearance mechanisms. The low dose is a factor of 10 above the TLV (treshold limit value) for mineral oil mistes, no untoward effects were expected at this level.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: daily (except weekends)DETAILED CLINICAL OBSERVATIONS: No BODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: NoFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data:No WATER CONSUMPTION: No OPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: Yes- How many animals: all core animals- Parameters examined: complete blood count (CBC) (white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and platelets) and differential count.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: Yes- How many animals: all core animals - Parameters examined: glucose, urea nitrogen, total protein, albumin (A), globulin (G), A/G, sorbitol dehydrogenase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total bilirubin, creatinine colesterol, triglycerides, uric acid, Cl, Ca, Na, K, and PURINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: NoOTHER:Lung function: The animals were anaesthetized and pulmonray function tests were performed (deflation quasistatic pressure-volume cureved, functional residual capactiy, and maximal forced exhalation maneuver). After the pulmonray function tests, the lungs were removed and all lobes were weighed. Lobes were frozen for analysis of hydroxyproline content and analysis of test material remaining in the lung.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; organ weights (adrenals, kidney, spleen, brain, liver, testes, epididymides, ovaries, thymus, heart, prostate, uterus, right middle lung lobeHISTOPATHOLOGY: Yes (untreated and high-dose): adrenals, ovaries, sternum, pancreas, brain, salivary gland, eye, spleen heart, stomach, colon, testes, duodenum, thymus, kidneys, thyroid, liver, tracheobronchial lymph nodes, lung, nasal turbinates, thigh muscle, urinary bladder, sciatic nerve, and any gross lesions. Only the lungs and tracheobronchial lymph nodes of the untreated controls were processed. 10 males of group 1, 2 and 5 (untreated, sham-exposed, and high-dose) were evaluated for morphology, number of sperm and number of testicular spermatids.

Statistics:

ANOVA and Tukey´s multiple range test: body weighs, male reproductive endpoints, haematology, and serum chemistryANOVA and Duncan´s multiple range test: organ weights, pulmonary function, and pulmonary hydroxyproline

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed.

Mortality:

no mortality observed

Description (incidence):

No mortalities observed.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Increased body weights were observed in treated males. The difference compared to control was statistically significant, but as no clear dose-response was seen and the difference was lower than 7%, it was not considered to be of toxicological relevance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The lungs had a minimal increase in weight after exposure to 0.50 mg/L. Other organ weights were not affected by exposure to the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The number of macrophages in the pulmonary alveoli increased slightly. This increase was small considering the high (500 mg/nr) aerosol concentration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of the lungs of animals in the high-dose group revealed one to two plump macrophages with sparse cytoplasmic vacuoles in less than 1.0% of the aveoli (in controls less than 0.1% would be expected). OTHER FINDINGS- Sperm morphology: No treatment-related effects were noted in sperm morophology or in sperm and spermatid counts. - Lung function: There were no significant differences between any groups for any of the pulmonary function parameters. The only parameter affected by exposure was the total weight of the five lung lobes. Weight for the high-dose group was significantly greater than the other groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEC

Effect level:

0.5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

0.5 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Jul - 10 Oct 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no data on test substance purity; only 2 dose groups, open application, limited parameters examined)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

(no data on test substance purity, only 2 dose groups, open application, limited parameters examined)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River, Lakeview, NJ, USA- Age at study initiation: approx. 7 weeks- Housing: individually in hanging, stainless steel cages with wire bottoms and fronts- Diet: Purina Certified Lab Chow ' 5002 in pellet form; ad libitum- Water: tap water; ad libitum- Acclimation period: at least 14 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20-22 - Humidity (%): 40-60- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE- Area of exposure: no data- Type of wrap if used: no wrap used, open- Time intervals for shavings or clipplings: 24 h before the first treatment; at least weekly afterwards- Application site: back (shaved)REMOVAL OF TEST SUBSTANCE- Washing (if done): no washing; wiping off with a gauze pads every saturday (applications on working days)TEST MATERIAL- Amount(s) applied (volume or weight with unit): no data - Concentration (if solution): undiluted- Constant volume or concentration used: noUSE OF RESTRAINERS FOR PREVENTING INGESTION: yes (collars), removal during the weekend

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days per week (65 exposures), 24 hours/day, removal of substance on saturdays (once a week)

Remarks:

Doses / Concentrations:800 and 2000 mg/kg bw/dayBasis:nominal per unit body weight

No. of animals per sex per dose:

10(5 additional animals of the control and the high dose group were included for dermal bioavailability experiments only.)

Control animals:

yes, concurrent no treatment

Details on study design:

The test substance was dispensed by volume from a syringe and left uncovered on the shaved skin. The rats were fitted with cardboard Elizabethan collars to minimze ingestion of the test material. The controls were treated in the same manner except that no material was applied to their skin.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: daily- Cage side observations included: appearance, behaviour, secretory function and dischargesDETAILED CLINICAL OBSERVATIONS: NoDERMAL IRRITATION (if dermal study): Yes- Time schedule for examinations: weekly- Parameters evaluated: erythema and edema according to Draize, chronic deterioration: flaking, thickening, stiffening, cracking and slouthingBODY WEIGHT: Yes - Time schedule for examinations: weeklyFOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: NoFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: NoWATER CONSUMPTION: NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: No data- How many animals: all animals- Parameters examined: red blood cells, white blood cells, and plateletsCLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: No data- How many animals: all animals- Parameters examined: glucose, urea nitrogen, alanine aminotransferase, albumin, phosphorus; only females: lactate dehydrogenase, aspartate aminotransferaseURINALYSIS: Yes- Time schedule for collection of urine: weeks 5 and 13- Metabolism cages used for collection of urine: No- Parameters examined: pH, bilirubin, specific gravity, urobilinogen, blood, protein, glucose, ketonesNEUROBEHAVIOURAL EXAMINATION: NoOTHER: sperm morphology: at termination- Parameters: percentage normal sperm, abnormal heads, headless, tailless, and curled tail

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; organ weights: kidneys, liver; only males: brain, spleen; only females: thyroidsHISTOPATHOLOGY: Yes (no further information available)

Statistics:

The level of statistical significance was P < 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Local effects: Slight erythema and flaking of the skin were observed in the treated groups during the dosing phase. Microscopic examination of the skin indicated trace to slight epidermal hyperplasia and chronic inflammation of the superficial dermis.

Mortality:

no mortality observed

Description (incidence):

No treatment-related effects were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treated males gained slightly less weight than the controls (800 mg/kg bw: 7%, 2000 mg/kg bw: 10%). As the difference is low, the decrease in body weight was not interpreted as a sign for systemic toxicity.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No adverse effects on any haematologic parameters measured were observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few of the serum parameters of the high-dose animals were statistically different from the controls, but the differences were small, not consistent between males and females, and did not present any pattern suggestive of effects on any specific organ (no corresponding histological findings). Thus, the effects were considered not to be of toxicological relevance. - high-dose males (compared to controls): glucose: -14%, albumin: -3%, and phoshorus: +16%- high-dose females (compared to controls): lactate dehydrogenase: +45% (low-dose: +22%), and aspartate aminotransferase: +22%.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No additional data given on Urinalysis in study report.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Increased thyroid weight in the low-dose (+ 25%) females and decreased spleen weight (- 10%) in the low-dose males were not considered to be toxicologically relevant, as these effects were not observed in the high-dose groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No abnormalities were detected.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

OTHER FINDINGS- Sperm morphology: No effects on sperm morphology were detected.
SKIN PENETRATION
Skin penetration values of 2 - 6% were obtained. The results of the in vivo skin penetration study indicate that the 13-week treatment with the test substance does not increase the skin penetration of the test substance (only the value for females was statistically different from the penetration in untreated animals). The skin penetration of untreated rats was less than 2% and the mean value for treated rats was approx. 6%. The recovery of radioactivity was measured in the urine and faeces as well as the remaining radioactivity in tissue samples.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Jul - 10 Oct 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no data on test substance purity; only 2 dose groups, open application, limited parameters examined)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

(no data on test substance purity, only 2 dose groups, open application, limited parameters examined)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River, Lakeview, NJ, USA- Age at study initiation: approx. 7 weeks- Housing: individually in hanging, stainless steel cages with wire bottoms and fronts- Diet: Purina Certified Lab Chow ' 5002 in pellet form; ad libitum- Water: tap water; ad libitum- Acclimation period: at least 14 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20-22 - Humidity (%): 40-60- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE- Area of exposure: no data- Type of wrap if used: no wrap used, open- Time intervals for shavings or clipplings: 24 h before the first treatment; at least weekly afterwards- Application site: back (shaved)REMOVAL OF TEST SUBSTANCE- Washing (if done): no washing; wiping off with a gauze pads every saturday (applications on working days)TEST MATERIAL- Amount(s) applied (volume or weight with unit): no data - Concentration (if solution): undiluted- Constant volume or concentration used: noUSE OF RESTRAINERS FOR PREVENTING INGESTION: yes (collars), removal during the weekend

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days per week (65 exposures), 24 hours/day, removal of substance on saturdays (once a week)

Remarks:

Doses / Concentrations:800 and 2000 mg/kg bw/dayBasis:nominal per unit body weight

No. of animals per sex per dose:

10(5 additional animals of the control and the high dose group were included for dermal bioavailability experiments only.)

Control animals:

yes, concurrent no treatment

Details on study design:

The test substance was dispensed by volume from a syringe and left uncovered on the shaved skin. The rats were fitted with cardboard Elizabethan collars to minimze ingestion of the test material. The controls were treated in the same manner except that no material was applied to their skin.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: daily- Cage side observations included: appearance, behaviour, secretory function and dischargesDETAILED CLINICAL OBSERVATIONS: NoDERMAL IRRITATION (if dermal study): Yes- Time schedule for examinations: weekly- Parameters evaluated: erythema and edema according to Draize, chronic deterioration: flaking, thickening, stiffening, cracking and slouthingBODY WEIGHT: Yes - Time schedule for examinations: weeklyFOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: NoFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: NoWATER CONSUMPTION: NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: No data- How many animals: all animals- Parameters examined: red blood cells, white blood cells, and plateletsCLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: at study termination- Animals fasted: No data- How many animals: all animals- Parameters examined: glucose, urea nitrogen, alanine aminotransferase, albumin, phosphorus; only females: lactate dehydrogenase, aspartate aminotransferaseURINALYSIS: Yes- Time schedule for collection of urine: weeks 5 and 13- Metabolism cages used for collection of urine: No- Parameters examined: pH, bilirubin, specific gravity, urobilinogen, blood, protein, glucose, ketonesNEUROBEHAVIOURAL EXAMINATION: NoOTHER: sperm morphology: at termination- Parameters: percentage normal sperm, abnormal heads, headless, tailless, and curled tail

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; organ weights: kidneys, liver; only males: brain, spleen; only females: thyroidsHISTOPATHOLOGY: Yes (no further information available)

Statistics:

The level of statistical significance was P < 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Local effects: Slight erythema and flaking of the skin were observed in the treated groups during the dosing phase. Microscopic examination of the skin indicated trace to slight epidermal hyperplasia and chronic inflammation of the superficial dermis.

Mortality:

no mortality observed

Description (incidence):

No treatment-related effects were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treated males gained slightly less weight than the controls (800 mg/kg bw: 7%, 2000 mg/kg bw: 10%). As the difference is low, the decrease in body weight was not interpreted as a sign for systemic toxicity.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No adverse effects on any haematologic parameters measured were observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few of the serum parameters of the high-dose animals were statistically different from the controls, but the differences were small, not consistent between males and females, and did not present any pattern suggestive of effects on any specific organ (no corresponding histological findings). Thus, the effects were considered not to be of toxicological relevance. - high-dose males (compared to controls): glucose: -14%, albumin: -3%, and phoshorus: +16%- high-dose females (compared to controls): lactate dehydrogenase: +45% (low-dose: +22%), and aspartate aminotransferase: +22%.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No additional data given on Urinalysis in study report.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Increased thyroid weight in the low-dose (+ 25%) females and decreased spleen weight (- 10%) in the low-dose males were not considered to be toxicologically relevant, as these effects were not observed in the high-dose groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No abnormalities were detected.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

OTHER FINDINGS- Sperm morphology: No effects on sperm morphology were detected.
SKIN PENETRATION
Skin penetration values of 2 - 6% were obtained. The results of the in vivo skin penetration study indicate that the 13-week treatment with the test substance does not increase the skin penetration of the test substance (only the value for females was statistically different from the penetration in untreated animals). The skin penetration of untreated rats was less than 2% and the mean value for treated rats was approx. 6%. The recovery of radioactivity was measured in the urine and faeces as well as the remaining radioactivity in tissue samples.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/cm²

Study duration:

subchronic

Species:

rat

Quality of whole database:

K1

Additional information

Repeated dose toxicity: oral

HATCOL 3331

Subacute 28-day oral toxicity with HATCOL 3331 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs daily; functional observation tests; bodyweight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Accuracy, homogeneity and stability over 4 hours of formulations test substance in propylene glycol were demonstrated by analyses.

Treatment-related findings observed were as follows:

50 mg/kg/day:- Renal hyaline droplets (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (1/5 males).

150 mg/kg/day:- Renal hyaline droplets (all males), increased incidence and severity of tubular basophilia in the kidneys (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (4/5 males).

1000 mg/kg/day:- Slightly lower weight gain on day 8 (males).

-lncreased absolute and relative food intake in weeks 1/2 (males).

-Slightly increased red blood cell count and haematocrit levels (females).

-lncreased liver weights and liver to body weight ratios (females).

-Renal hyaline droplets: (all males), increased incidence and severity of tubular basophilia in the kidneys (3/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (all males).

From the results presented in the report a No Observed Adverse Effect Level (NOAEL) could not be determined for males, but was established to be 150 mg/kg/day for females. However, since the male kidney findings are a species and sex specific response which is not observed in humans, a NOAEL of 150 mg/kg/day may be considered for both sexes.

 

HATCOL 3344

Subacute 28-day oral toxicity with HATCOL 3344 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Accuracy and homogeneity of formulations of test substance in propylene glycol were acceptable. Group 2 formulations (50 mg/kg/day) were not stable over a 4-hour period (approximately 50% recovery), while stability of group 4 formulations (1000 mg/kg/day) was confirmed over the same time-period. Based on overall results and the established NOAEL, it was considered that sufficient analytical support was obtained for the purpose of this study.

Treatment-related findings observed were as follows:

50 mg/kg/day: No treatment-related findings noted.

150 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: No treatment-related findings noted.

CONCLUSION

Based on the results presented in this report, a NOAEL of 1000 mg/kg/day for HATCOL 3344 was established.

 

HATCOL 5236

Subacute 28-day oral toxicity with HATCOL 5236 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.  

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Accuracy, homogeneity and stability over at least 1 hour (50 mg/kg formulations) or at least 2 hours (1000 mg/kg formulations) of formulations of test substance in propylene glycol were demonstrated by analyses.

50 mg/kg/day: No treatment-related findings noted.

150 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: Higher red blood cell count (F), lower cholesterol values(M).

Subacute 28-day oral toxicity with HATCOL 5236 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.  

CONCLUSION

The higher red blood cell count and the lower cholesterol values found in the high dose females and males respectively, were not supported by other changes in blood parameters or histopathological lesions. Therefore, the toxicological relevance of these effects is doubtful. An enlarged of the liver was noted in two males at 1000 mg/kg/day. Since this was not seen in the other high dose animals and since no morphological correlates were found, this finding was considered not to be toxicologically relevant.

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for HATCOL 5236 of 150 mg/kg/day was established. Based on the absence of functional or morphological disturbances supporting higher red blood cell count and lower cholesterol values in the high dose females and males respectively, a NOAEL of 1000 mg/kg/day may be considered.

 

HATCOL 1765

A 28 day repeated dose toxicity study by the oral route was conducted with pentaerythritol ester, tetrasubstituted (CAS 68424-31-7). The doses administered were 0 ppm, 1000 ppm, 5000 ppm, 12500 ppm (nominal in the diet). Repeated dietary administration of the test material to rats, up to and including a dose level of 1450 mg/kg bw/day for male rats and 1613 mg/kg bw/d for female rats, did not produce any evidence of overt toxicity. There were no clinical signs indicative of neurological dysfunction or neuropathological changes in the brain related to treatment with the test material at any dose level. Treatment related histopathological changes in the kidney (including increased tubular hyaline droplet formation and tubular basophilia) and changes in kidney and liver weight in male rats at 5000 ppm and above were considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination. The NO(A)EL for the read-across substance was therefore considered to be 12500 ppm in males (actual dose received: 1450 mg/kg/day) and females (actual dose received: 1613 mg/kg/day).

 

CAS 403507-18-6

A 90 day repeated dose toxicity study by the oral route was conducted with CAS 403507-18-6; the substance was administered at doses of 5, 50, and 1000 mg/kg bw/day to male/female Sprague-Dawley rats. There were no treatment-related deaths during the 90-day study and no treatment-related effects on clinical chemistry and haematological parameters, body weight and organ weight. Increased salivation was detected immediately after dosing for 1000 mg/kg bw/day females from Day 17 and for 1000 mg/kg bw/day males from Day 18 onwards. Hunched posture was observed for individual 1000 mg/kg bw/day females from Day 37 with isolated incidents of tiptoe gait also observed on Days 37 and 38 and exophthalmia present in two females from Day 75 and 76 onwards. Exophthalmia was also evident in two 50 mg/kg bw/day females from Day 81 and hunched posture was observed for another 50 mg/kg bw/day female from Day 81 onwards. No such observations were detected for animals of either sex treated with 5 mg/kg bw/day.On necropsy, no treatment-related macroscopic abnormalities and no treatment-related histopathological changes were detected. The NOAEL was therefore determined to be ≥ 1000 mg/kg/day.

 

Repeated does toxicity: inhalation

CAS 67762-53-2

A 90 day repeated dose toxicity study by the inhalation route was conducted with CAS 67762-53-2; the substance was administered at doses of 0.05, 0.15, and 0.5 mg/L to male/female Sprague-Dawley rats.The NOAEC was therefore determined to be 0.5 mg/L air. No treatment-related effects were observed.

 

Repeated dose toxicity: dermal

CAS 67762-53-2

A 90 day repeated dose toxicity study by the dermal route was conducted with CAS 67762-53-2; the substance was administered at doses of 800 and 2000 mg/kg bw/day to male/female Sprague-Dawley rats.The NOAEL was therefore determined to be ≥ 2000 mg/kg/day. No toxicologically relevant symptoms/signs occurred; treated males gained slightly less weight than the controls (800 mg/kg bw: 7%, 2000 mg/kg bw: 10%). As the difference is low, the decrease in body weight was not interpreted as a sign for systemic toxicity.Sight erythema and flaking; slight epidermal hyperplasia and chronic inflammation occurred in both treatment groups.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Effect level derived by experiment to OCD guideline 407, EU test standard B7 and ES EPA procedure 870.3050