Benzoic acid, 2-hydroxy-, C14-18-alkyl derivs.

Endpoint summary

Description of key information

ORAL
28 Day systemic NOAEL 100 mg/kg (male and female), rat; OECD 407, EU Method B.7 and US EPA OPPTS 870.3050

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September 2014 to 03 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation (mean): Males: 145 g; Females: 122 g
- Fasting period before study: No, though during motor activity measurements animals had no access to food.
- Housing: Group housing of up to 5 animals per sex in cages (height 18 cm) with sterilised sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: ad libitum pelleted rodent diet
- Water: Free access to tap water except during motor activity measurements
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: No data
To: 03 November 2014

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were stirred during dosing of all animals. Adjustment was made for specific gravity of the vehicle.

DOSE VOLUME: 5 mL/kg bw

VEHICLE
- Specific gravity: 1.036

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Samples of dose preparations were stored and dispatched on dry ice to the test site for formulation analysis.
Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90 to 110 % of target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.
Additionally, the long-term stability of the formulation samples prepared in advance of the study and stored at a target temperature =-70 °C was determined.

SAMPLES
In total, 16 samples were included in this study, distributed over 4 formulation groups. The samples of the control group and the 100 mg/kg dose group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 30 and 300 mg/kg were taken in duplicate from the top, middle and bottom position of the container.
The samples were stored at a target temperature =-70 °C.

ANALYTICAL METHOD
- Analytical conditions
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
- Sample injections
The analytical run for the determination of the accuracy of preparation and homogeneity of the test material in formulations was acquired in the following order: analytical blank sample, calibration curve, analytical blank sample, analytical blank sample, procedural recovery sample high and low (replicate 1), analytical samples, long term storage stability samples and procedural recovery samples high and low (replicate 2).
The reinjection of the analytical run for the determination of the accuracy of preparation, homogeneity of the test material in formulations and storage stability was acquired in the following order: analytical blank sample (fresh calibration curve), calibration curve (fresh calibration curve), procedural recovery sample high and low fresh (replicate 1), procedural recovery samples high and low fresh (replicate 2), analytical blank sample, calibration curve, analytical blank sample, analytical blank sample, procedural recovery sample high and low (replicate 1), analytical samples, long term storage stability samples and procedural recovery samples high and low (replicate 2).
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAS
Response (Y): Peak area test material [mAU]

Calibration curve: Y = a + bX + cX²
where:
X = nominal concentration [mg/L]
c = curvature
b = slope
a = intercept

Analysed concentration (X): X = [-b + v(b² - 4c(a – y)) / 2c] x ((V x d) / w) [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
[-b + v(b² - 4c(a – y)) / 2c] values are obtained from Analyst® version 1.5.2 (Applied Biosystems, Burlington, Canada).

Recovery: (Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy: (Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.): ((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
- Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted. Outliers could be discarded as long as minimally 75 % of the responses of the calibration were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 90 to 110 %.
The response in all analytical blank samples at retention time of the test material should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
- Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %. Formulations were considered to be stable if the relative difference between the stored and freshly taken samples was =10 %.

RESULTS
- Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Regression analysis was performed using the least squares quadratic regression with a (1 / concentration²) weighting factor. The coefficient of correlation (r) was always higher than or equal to 0.9997.
No response at the retention time of the test material in the analytical blank samples was detected.
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
- Test samples
In the control group formulations, no test material was detected. The concentrations analysed in the formulations of the dose groups were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %).
The formulations of the 30 and 300 mg/kg dose groups were homogeneous (i.e. coefficient of variation = 10 %).
- Stability at storage conditions
The mean accuracies were 101.0 and 93.5 % for the high (200 mg/g) and the low (1.00 mg/g) concentration levels, respectively, which are within the set criteria (mean accuracy is in the range 90 to 110 %). It was therefore concluded that the test material was stable in propylene glycol over a storage period of at least 56 days at a temperature of =-70 °C.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a 14 day range-finding study in which the rats were dosed at 500 and 1000 mg/kg bw.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality and viability were made at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from the start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing and between 1 and 2 hours (± 30 minutes) after dosing (based on peak effect of occurrence of clinical signs observed in the dose range finding study). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected on the day of necropsy at the end of the treatment phase. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals
- Haematology parameters examined: White blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils and basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets.
- Clotting parameters examined: Prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected on the day of necropsy at the end of the treatment phase. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals
- Parameters examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorganic phosphate (Inorg. Phos).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 4 of treatment, the following tests were performed on all animals after dosing between 1 and 2 hours after dosing, except for the motor activity measurement (based on the peak effect in occurrence of clinical signs in the dose range finding study).
- Dose groups that were examined: All
- Battery of functions tested: hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R), fore- and hind-limb grip strength (recorded as the mean of three measurements, Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands) and locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerised monitoring system, Kinder Scientific LLC, Poway, USA).

Sacrifice and pathology:

SACRIFICE
On the scheduled day of necropsy, animals were deeply anaesthetised using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied.

PATHOLOGY
Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution): Adrenal glands, (aorta), brain [cerebellum, mid-brain, cortex], caecum, cervix, (clitoral gland), colon, duodenum, epididymides*, eyes (including 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 including coagulating gland, skeletal muscle, (skin), spinal cord [cervical, midthoracic, lumbar], spleen, sternum with bone marrow, stomach, testes*, thymus, thyroid including parathyroid [if detectable], (tongue), trachea, urinary bladder, uterus, vagina and all gross lesions.
Tissues marked with * were fixed in modified Davidson's solution; tissues were transferred to formalin after fixation for at least 24 hours.
Tissues/organs mentioned in parentheses were not examined by the pathologist.

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, ovaries, spleen, testes, thymus, uterus (including cervix), prostate*, seminal vesicles including coagulating glands* and thyroid including parathyroid.
Organs marked with * were weighed when fixed for at least 24 hours.

HISTOTECHNOLOGY
All organ and tissue samples were processed, embedded in paraffin wax, cut at a thickness of 2 to 4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
The following slides were examined:
- All tissues collected at the scheduled sacrifice from all control and high dose animals;
- Stomach (forestomach)and liver of all low and mid-dose animals based on (possible) treatment-related changes in these organs in the high dose group;
- All gross lesions.

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 if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
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. Test statistics were calculated on the basis of exact values for means and pooled variances.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

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):

see below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
Salivation was noted after dosing at 100 and 300 mg/kg/day starting from Week 1 or 2 onwards. In addition, a few males at 300 mg/kg/day showed hunched posture and/or piloerection only on a few days and females at this dose level showed hunched posture at an even lower incidence.
No further treatment related clinical signs were noted during detailed daily observations and weekly arena observations.
Other clinical signs noted during the treatment period occurred within the range of background findings for rats of this age and strain which are housed and treated under the conditions used in this study and did not show any apparent dose-related trend.

BODY WEIGHT
Body weights and body weight gain of males treated at 300 mg/kg/day were significantly lower than controls throughout the treatment period.
No changes were noted in body weights and body weight gains of the other treated animals.

FOOD CONSUMPTION
Food consumption of males at 300 mg/kg/day was lower compared to controls during the study period.
However the food consumption relative to body weight recovered to normal within 2 weeks.
No changes in food consumption before or after correction for body weight were noted in the other treated animals.

HAEMATOLOGY
The following statistically significant changes in haematology parameters were noted:
- Higher white blood cell counts (WBC) in males and females at 300 mg/kg/day; and
- Lower relative eosinophil counts (%WBC) in males at 300 mg/kg/day.
Other statistically significant changes in haematological parameters occurred in the absence of a dose-related trend, and were therefore considered to be unrelated to treatment.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters were noted:
- Higher alanine aminotransferase activity (ALAT) in males and females at 300 mg/kg/day (at 100 mg/kg/day alanine aminotransferase activity appeared higher on an individual animal basis);
- Higher aspartate aminotransferase activity (ASAT) in males and females at 300 mg/kg/day;
- Higher alkaline phosphatase activity (ALP) in males and females at 300 mg/kg/day (with an increasing trend at lower dose levels);
- Lower total protein level in males and females at 300 mg/kg/day (not statistically significant in females);
- Higher potassium in males and females at 300 mg/kg/day (not statistically significant in males);
- Lower chloride in females at 300 mg/kg/day; and
- Higher inorganic phosphate in females at 300 mg/kg/day.
Other statistically significant differences in clinical biochemistry parameters showed no dose-response related trends and were therefore considered not treatment related.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

GROSS PATHOLOGY
Irregular surface of the forestomach was noted for all males and females at 300 mg/kg/day.
The incidence of other necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be unrelated to treatment with the test material.

ORGAN WEIGHTS
Higher absolute and/or relative liver weights were noted for females at 100 mg/kg/day and for males and females at 300 mg/kg/day (relative liver weights were increased by 9, 17 and 23 %, respectively, when compared to mean control levels).
Other statistically significant organ weight differences were considered to be the result of a lower terminal body weight and/or were considered not related to treatment based on no clear dose-response trend and no microscopic correlates.

HISTOPATHOLOGY
Findings in the forestomach were observed in females treated at 100 mg/kg/day and males and females treated at 300 mg/kg/day and consisted of:
- Granulocytic subepithelial inflammation in 1/5 females (minimal) at 100 mg/kg/day and in 5/5 males (2 minimal, 3 slight) and 5/5 females (2 minimal, 3 slight) at 300 mg/kg/day;
- Hyperplasia of the squamous epithelium in 1/5 females (slight) at 100 mg/kg/day and in 5/5 males (3 minimal, 2 slight) and 5/5 females (4 minimal, 1 slight) at 300 mg/kg/day;
- Hyperkeratosis in 1/5 females (minimal) at 100 mg/kg/day and in 5/5 males (3 slight, 2 moderate) and 5/5 females (1 minimal, 2 slight, 2 moderate) at 300 mg/kg/day; and
- Submucosal oedema in 1/5 females (slight) at 100 mg/kg/day and in 2/5 males (1 minimal, 1 slight) and 2/5 females (1 minimal, 1 slight) at 300 mg/kg/day.
Findings in the liver were observed in females treated at 300 mg/kg/day and consisted of:
- Hepatocellular hypertrophy in 4/5 females (4 minimal).
There were no other test material-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

DISCUSSION AND CONCLUSION
No mortality occurred and no treatment-related changes in functional observation tests were observed.
Clinical observations revealed salivation in all males and females at 100 and 300 mg/kg/day, which could be correlated to the irritancy of the test material. Hunched posture and piloerection were noted at 300 mg/kg/day at a low incidence. In addition, at this dose level the body weight and food consumption were lower in males compared to control levels.
Along with these clinical observations, the histopathological examination revealed hyperkeratosis (up to moderate degree), subepithelial granulocytic inflammation (up to slight degree), hyperplasia of the squamous epithelium (up to slight degree) and/or submucosal oedema (up to slight degree) in the forestomach among males and females at 300 mg/kg/day. These findings were also noted in females at 100 mg/kg/day at a lower incidence and/or severity. These microscopic findings were considered to reflect damage of the forestomach epithelium by gavage with the test material as a bolus and therefore are regarded to be adverse in nature at the portal-of-entry.
At the highest dose level the higher white blood cell counts in males and females may be associated with the inflammatory responses seen in the stomach. The lower relative eosinophil level recorded in high dose males may be stress-related however, since the relative eosinophil levels were only marginally affected and remained well within the historical control range, this finding was not considered to be adverse.
Higher absolute and/or relative liver weights were observed in males and females at 300 mg/kg/day (relative weight, respectively, 17 and 23 % higher compared to controls) and at 100 mg/kg/day (relative weight 9 % higher compared to controls). The higher liver weight was associated to minimal degree of liver hepatocellular hypertrophy in females at 300 mg/kg/day. These morphological changes were further accompanied by changes in clinical biochemistry parameters (i.e. higher alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities and lower total protein). Although there were no adverse morphological findings in the liver, the higher liver enzyme activities and the magnitude of increase in liver weight (max. 23 % higher relative weight) are considered to be toxicologically relevant and adverse in nature at 300 mg/kg/day. At 100 mg/kg/day, the minimal increase in liver weight was considered not adverse in nature since not accompanied by any microscopic findings or concurrent toxicologically relevant changes in clinical biochemistry parameters.
No clear morphological correlates were noted for changes in clinical biochemistry, such as higher potassium and inorganic phosphate levels and lower chloride levels. In addition, the changes were slight in nature and therefore considered not to be adverse.

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on the morphologic changes in the forestomach, consisting of hyperkeratosis, hyperplasia of the squamous epithelium, submucosal oedema, and subepithelial granulocytic inflammation.

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on the morphologic changes in the forestomach, consisting of hyperkeratosis, hyperplasia of the squamous epithelium, submucosal oedema, and subepithelial granulocytic inflammation.

Dose descriptor:

NOAEL

Remarks:

- systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects on the liver, consisting of increases in weight and liver enzyme activities .

Critical effects observed:

not specified

Table 1: Summary of Body Weights (g)

Day of Measurement		Males				Females
		Dose Group (mg/kg)
		0	30	100	300	0	30	100	300
1	Mean SD N	145 6.0 5	146 3.2 5	145 8.4 5	143 4.9 5	121 6.6 5	122 6.1 5	122 4.5 5	122 6.7 5
8	Mean SD N	194 6.1 5	196 1.5 5	192 8.6 5	175** 7.1 5	144 5.8 5	148 7.9 5	147 7.0 5	142 7.3 5
15	Mean SD N	245 11.2 5	248 2.8 5	239 10.0 5	216** 9.5 5	167 7.8 5	169 6.5 5	173 6.1 5	170 11.1 5
22	Mean SD N	275 12.4 5	283 6.2 5	271 8.7 5	244** 9.6 5	183 8.7 5	188 7.0 5	189 9.4 5	186 13.6 5
28	Mean SD N	303 15.7 5	310 12.5 5	299 9.4 5	268** 11.7 5	193 10.5 5	196 12.6 5	198 11.5 5	198 15.9 5

**Significant at the 1 % level using the Dunnett-test based on pooled variance

 

Table 2: Summary of Selected Clinical Chemistry Parameters

Parameter		Males				Females
		Dose Group (mg/kg)
		0	30	100	300	0	30	100	300
ALAT (U/L)	Mean SD N	54.9 15.5 5	44.4 9.0 5	74.6 13.0 5	152.3** 29.5 5	40.3 7.0 5	41.4 13.7 5	55.9 20.4 5	113.2** 38.2 5
ASAT (U/L)	Mean SD N	77.0 11.5 5	68.3 3.9 5	85.5 13.0 5	93.2* 5.4 5	68.3 6.4 5	70.6 5.1 5	77.7 11.0 5	91.3** 14.6 5
ALP (U/L)	Mean SD N	242 54 5	254 35 5	377 119 5	624** 306 5	93 28 5	111 46 5	177 54 5	315** 187 5
Total Protein (g/L)	Mean SD N	64.0 1.9 5	63.1 1.4 5	62.2 2.0 5	60.5* 1.7 5	64.0 2.0 5	63.3 2.8 5	64.2 1.6 5	61.6 2.0 5
Glucose (mmol/L)	Mean SD N	7.91 0.6 5	8.97 0.51 5	9.67* 1.10 5	9.13 1.33 5	6.41 0.51 5	6.64 0.42 5	7.24 0.99 5	6.97 0.78 5
Potassium (mmol/L)	Mean SD N	3.85 0.20 5	3.87 0.15 5	3.99 0.07 5	4.05 0.11 5	3.46 0.18 5	3.50 0.34 5	3.59 0.25 5	4.04** 0.10 5
Chloride (mmol/L)	Mean SD N	100 1 5	99 1 5	100 2 5	99 1 5	103 1 5	103 1 5	103 1 5	101* 1 5
Calcium (mmol/L)	Mean SD N	2.69 0.06 5	2.65 0.02 5	2.57** 0.04 5	2.66 0.05 5	2.58 0.10 5	2.58 0.04 5	2.58 0.07 5	2.60 0.03 5
Inorganic Phosphate (mmol/L)	Mean SD N	2.92 0.25 5	2.87 0.34 5	2.89 0.21 5	2.61 0.27 5	1.99 0.17 5	2.10 0.23 5	1.83 0.27 5	2.46* 0.21 5

ALAT = Alanine aminotransferase

ASAT = Aspartate aminotransferase

ALP = Alkaline phosphatase

*Significant at the 5 % level using the Dunnett-test based on pooled variance

**Significant at the 1 % level using the Dunnett-test based on pooled variance

 

Table 3: Selected Absolute and Relative Organ Weights

Parameter	Unit		Males				Females
			Dose Group (mg/kg)
			0	30	100	300	0	30	100	300
Body	g	Mean SD N	278 14 5	288 8 5	273 10 5	243* 10 5	177 8 5	181 11 5	186 5 5	178 14 5
Brain	g	Mean SD N	1.97 0.09 5	1.96 0.05 5	1.92 0.08 5	1.97 0.08 5	1.83 0.07 5	1.85 0.06 5	1.86 0.08 5	1.83 0.04 5
	%	Mean SD N	0.71 0.04 5	0.68 0.04 5	0.70 0.01 5	0.81** 0.02 5	1.04 0.03 5	1.03 0.04 5	1.00 0.04 5	1.03 0.08 5
Liver	g	Mean SD N	8.08 0.38 5	8.84 0.83 5	8.50 0.49 5	8.30 0.43 5	4.90 0.32 5	5.32 0.34 5	5.64** 0.29 5	6.09** 0.27 5
	%	Mean SD N	2.91 0.05 5	3.07 0.21 5	3.11 0.10 5	3.41** 0.10 5	2.78 0.09 5	2.94 0.12 5	3.03* .011 5	3.43** 0.19 5
Kidneys	g	Mean SD N	2.06 0.08 5	2.13 0.15 5	2.17 0.09 5	1.83** 0.05 5	1.42 0.08 5	1.49 0.09 5	1.52 0.10 5	1.53 0.16 5
	%	Mean SD N	0.74 0.04 5	0.74 0.04 5	0.80 0.02 5	0.75 0.03 5	0.81 0.03 5	0.82 0.05 5	0.82 0.04 5	0.86 0.06 5
Adrenals	g	Mean SD N	0.054 0.008 5	0.062 0.008 5	0.060 0.008 5	0.060 0.008 5	0.065 0.008 5	0.066 0.011 5	0.062 0.017 5	0.065 0.008 5
	%	Mean SD N	0.020 0.003 5	0.022 0.002 5	0.022 0.002 5	0.025* 0.004 5	0.037 0.004 5	0.037 0.005 5	0.033 0.009 5	0.037 0.006 5
Spleen	g	Mean SD N	0.491 0.065 5	0.524 0.076 5	0.559 0.049 5	0.455 0.047 5	0.353 0.028 5	0.416* 0.034 5	0.401 0.041 5	0.361 0.043 5
	%	Mean SD N	0.177 0.025 5	0.182 0.025 5	0.205 0.019 5	0.187 0.021 5	0.200 0.011 5	0.230 0.017 5	0.215 0.019 5	0.204 0.030 5
Testes	g	Mean SD N	3.18 0.22 5	3.16 0.15 5	3.32 0.26 5	3.21 0.17 5	-	-	-	-
	%	Mean SD N	1.15 0.11 5	1.10 0.03 5	1.21 0.07 5	1.32** 0.09 5	-	-	-	-
Prostate	g	Mean SD N	0.452 0.073 5	0.306** 0.054 5	0.365 0.068 5	0.275** 0.035 5	-	-	-	-
	%	Mean SD N	0.163 0.027 5	0.107** 0.020 5	0.134 0.027 5	0.113** 0.013 5	-	-	-	-
Seminal vesicles	g	Mean SD N	0.764 0.119 5	0.736 0.116 5	0.778 0.130 5	0.453** 0.106 5	-	-	-	-
	%	Mean SD N	0.276 0.048 5	0.256 0.045 5	0.285 0.049 5	0.187* 0.047 5	-	-	-	-

*Significant at the 5 % level using the Dunnett-test based on pooled variance

**Significant at the 1 % level using the Dunnett-test based on pooled variance

Conclusions:

Under the conditions of this study the local NOAEL was determined to be 30 and 100 mg/kg/day for females and males, respectively. The systemic NOAEL was determined to be 100 mg/kg/day for males and females.

Executive summary:

The repeated dose toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 407, EU Method B.7 and US EPA OPPTS 870.3050 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats at dose levels of 0, 30, 100 and 300 mg/kg/day. Five rats per sex per dose were treated and chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters were evaluated: mortality, clinical signs, functional observations, body weight, food consumption, haematology and clinical chemistry. At the end of the treatment period the rats were sacrificed and subjected to a complete necropsy including gross pathology and histopathology examinations.

Formulation analysis confirmed that formulations of the test material in propylene glycol were prepared accurately and were homogenously distributed.

No mortality occurred and no treatment-related changes in functional observation tests were observed.

Clinical observations revealed salivation in all males and females at 100 and 300 mg/kg/day, which could be correlated to the irritancy of the test material. Hunched posture and piloerection were noted at 300 mg/kg/day at a low incidence. In addition, at this dose level the body weight and food consumption were lower in males compared to control levels.

Along with these clinical observations, the histopathological examination revealed hyperkeratosis (up to moderate degree), subepithelial granulocytic inflammation (up to slight degree), hyperplasia of the squamous epithelium (up to slight degree) and/or submucosal oedema (up to slight degree) in the forestomach among males and females at 300 mg/kg/day. Similar findings were also noted in females at 100 mg/kg/day at a lower incidence and/or severity. The microscopic findings were considered to reflect damage of the forestomach epithelium by gavage with the test material as a bolus and therefore are regarded to be adverse in nature at the portal-of-entry.

At the highest dose level, the higher white blood cell counts in males and females may be associated with the inflammatory responses seen in the stomach.

Higher absolute and/or relative liver weights were observed in males and females at 300 mg/kg/day (relative weight, respectively, 17 and 23 % higher compared to controls) and at 100 mg/kg/day (relative weight 9 % higher compared to controls). The higher liver weight was noted along with a minimal degree of liver hepatocellular hypertrophy in females at 300 mg/kg/day. These morphological changes were further accompanied by changes in clinical biochemistry parameters (i.e. higher alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities and lower total protein). Although there were no adverse morphological findings in the liver, the higher liver enzyme activities and the magnitude of increase in liver weight (max. 23 % higher relative weight) are considered to be toxicologically relevant and adverse in nature at 300 mg/kg/day. At 100 mg/kg/day, the minimal increase in liver weight was considered not adverse in nature since not accompanied by any microscopic findings or concurrent toxicologically relevant changes in clinical biochemistry parameters.

No clear morphological correlates were noted for changes in clinical biochemistry, such as higher potassium and inorganic phosphate levels and lower chloride levels. In addition, the changes were slight in nature and therefore considered not to be adverse.

Under the conditions of this study the local NOAEL was determined to be 30 and 100 mg/kg/day for females and males, respectively. The systemic NOAEL was determined to be 100 mg/kg/day for males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was conducted in accordance with standardised guidelines under GLP conditions. It was awarded a reliability core of 1 in accordance with the criteria for assessing data quality as set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be good.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute Toxicity

The repeated dose toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 407, EU Method B.7 and US EPA OPPTS 870.3050 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats at dose levels of 0, 30, 100 and 300 mg/kg/day. Five rats per sex per dose were treated and chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters were evaluated: mortality, clinical signs, functional observations, body weight, food consumption, haematology and clinical chemistry. At the end of the treatment period the rats were sacrificed and subjected to a complete necropsy including gross pathology and histopathology examinations.

Formulation analysis confirmed that formulations of the test material in propylene glycol were prepared accurately and were homogenously distributed.

No mortality occurred and no treatment-related changes in functional observation tests were observed.

Clinical observations revealed salivation in all males and females at 100 and 300 mg/kg/day, which could be correlated to the irritancy of the test material. Hunched posture and piloerection were noted at 300 mg/kg/day at a low incidence. In addition, at this dose level the body weight and food consumption were lower in males compared to control levels.

Along with these clinical observations, the histopathological examination revealed hyperkeratosis (up to moderate degree), subepithelial granulocytic inflammation (up to slight degree), hyperplasia of the squamous epithelium (up to slight degree) and/or submucosal oedema (up to slight degree) in the forestomach among males and females at 300 mg/kg/day. Similar findings were also noted in females at 100 mg/kg/day at a lower incidence and/or severity. The microscopic findings were considered to reflect damage of the forestomach epithelium by gavage with the test material as a bolus and therefore are regarded to be adverse in nature at the portal-of-entry.

At the highest dose level, the higher white blood cell counts in males and females may be associated with the inflammatory responses seen in the stomach.

Higher absolute and/or relative liver weights were observed in males and females at 300 mg/kg/day (relative weight, respectively, 17 and 23 % higher compared to controls) and at 100 mg/kg/day (relative weight 9 % higher compared to controls). The higher liver weight was noted along with a minimal degree of liver hepatocellular hypertrophy in females at 300 mg/kg/day. These morphological changes were further accompanied by changes in clinical biochemistry parameters (i.e. higher alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities and lower total protein). Although there were no adverse morphological findings in the liver, the higher liver enzyme activities and the magnitude of increase in liver weight (max. 23 % higher relative weight) are considered to be toxicologically relevant and adverse in nature at 300 mg/kg/day. At 100 mg/kg/day, the minimal increase in liver weight was considered not adverse in nature since not accompanied by any microscopic findings or concurrent toxicologically relevant changes in clinical biochemistry parameters.

No clear morphological correlates were noted for changes in clinical biochemistry, such as higher potassium and inorganic phosphate levels and lower chloride levels. In addition, the changes were slight in nature and therefore considered not to be adverse.

Under the conditions of this study the local NOAEL was determined to be 30 and 100 mg/kg/day for females and males, respectively. The systemic NOAEL was determined to be 100 mg/kg/day for males and females.

Subchronic and Chronic Toxicity

In accordance with Section 8.6.2. of Column 2 of Annex IX of the REACH Regulation, the study does not need to be conducted if a reliable short-term toxicity study (28 days) is available showing severe toxicity effects according to the criteria for classifying the substance as R48, for which the observed NOAEL-28 days, with the application of an appropriate uncertainty factor, allows the extrapolation towards the NOAEL-90 days for the same route of exposure.

In such a study, conducted under GLP conditions to EU method B.7/OECD Guideline 407, the NOAEL of the substance was determined to be 100 mg/kg/day and the substance has been classified by the registrant as STOT RE Category 2 and is considered to show sufficient toxicity effects so as to meet the criteria for classifying the substance as R48. This study is considered to allow extrapolation towards the NOAEL-90 for the same route of exposure. As such the registrant proposes to waive the 90-Day Repeat Dose Toxicity study.

Furthermore, in accordance with Section 3 of Annex XI, the 90-Day Sub-Chronic Repeat Dose study may be omitted based on exposure scenarios developed in the chemical safety report, given that the comparison of the derived DNEL with the results of the exposure assessment shows that exposures are always well below the derived DNEL. The registrant considers that the grounds for waiving this study are strengthened due to the low potential for exposure to the substance throughout its lifecycle as demonstrated in the chemical safety report and that any potential extra information that could be gained from the performance of this study is outweighed by the opportunity to prevent unnecessary vertebrate animal testing.

Concerning chronic toxicity testing, the substance is not considered to fulfil any of the criteria to propose further testing under Section 8.6.3. of Column 2 of Annex X.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance requires classification with respect to specific target organ toxicity after repeated exposure as Category 2, H373: May cause damage to organs (liver).