Fatty acids, coco, esters with 1,3-butanediol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Oct 2006 - 09 May 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analytical purity stated, lack of historical control data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

yes

Remarks:

no analytical purity stated, lack of historical control data.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd: Sprague Dawley SD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l. (San Pietro al Natisone (UD), Italy)
- Age at study initiation: 9-10 weeks (females) and 11 weeks (males)
- Weight at study initiation: 202-223 g (females) and 335-342 g (males)
- Housing: Before and after pairing, the animals were housed in groups of no more than 5 per sex in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Varese, Italy).
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was suspended in the vehicle (corn oil) at a concentration of 500 mg/mL. The formulations were prepared daily and concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Concentration in vehicle: 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed to confirm that the proposed formulation procedure was acceptable. The stability of the formulations was found to be 24 h at room temperature. Samples of the formulations prepared during the first and last week of the study were analysed to check the concentration and homogeneity. Homogeneity and recovery were within the acceptable limits.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear or the presence of a copulation plug was referred to as Day 0 of pregnancy (Day 0 post coitum)
- Other: After proof of pregnancy, the female was separated from the male, which was paired with a new stock female. One male was used to mate with no more than three females.

Duration of treatment / exposure:

Day 6-19 post mating

Frequency of treatment:

daily, 7 days/week

Duration of test:

Day 20 post mating

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 P females

Control animals:

yes, concurrent vehicle

Details on study design:

- Route selection rationale: The oral route of administration was selected because it is a possible route of exposure of the test item in man.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, prior to dosing, approximately 30 min and 2 h after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 9, 12, 15 and 20 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: a detailed post mortem examination was conducted (including examination of the external surface and orifices, no further details)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- gross evaluation of placentae
- Number and distribution of intra-uterine deaths

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Other: number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing)

Statistics:

For continuous variables (body weight, body weight gain, food consumption), the significance of the difference amongst group means was assessed by analysis of variance. Differences between the treated group and the control group were assessed by Dunnett’s test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett’s test before Dunnett’s test was performed. If the data were found to be inhomogeneous, a modified t-test (Cochran and Cox) was applied. The criteria for statistical significance were p < 0.05 and 0.01. The non-parametric Kruskal-Wallis analysis of variance was used for all other parameters (gravid uterus weight, corrected body weight, litter data and sex ratio). Intergroup differences between the control and the treated group were assessed by the non-parametric version of the Williams' test using the SAS System Program. The criteria for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Indices:

Pre-implantation loss was calculated as a percentage from the formula: [(no. of corpora lutea - no. of implantations) / no. of corpora lutea] x 100

Post-implantation loss was calculated as a percentage from the formula: [(no. of implantations - no. of live young) / no. of implantations] x 100

Total implantation loss was calculated as a percentage from the formula: [(no. of corpora lutea - no. of live young) / no. of corpora lutea] x 100

Sex ratios of the foetuses were calculated as the percentage of males per litter.

All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No mortality occurred during the study. One treated female was found not pregnant at necropsy. The numbers of dams with live foetuses at necropsy were 24 in the control group and 23 in the treated group. No reactions to treatment were noted at pre- and post-dose observations. Animals did not show any treatment-related effects during clinical sign observations. Body weight, body weight gain and food consumption were unaffected by treatment. No treatment-related effects were seen in terminal body weight, uterus weight or absolute weight gain. No macroscopic changes were detected in treated females.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: overall effects

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
A slight but statistically significant lower mean foetal weight was observed in the treatment group when compared to the control group. This slight difference was attributed to the higher presence of foetuses in the treated group compared to controls in which mean foetal weight was also unusually higher when compared to the internal historical control data. The mean foetal weight of the treated group was well within the historical control data for this rat species (3.03 - 4.2 g, mean 3.6 g). Three small foetuses were present at necropsy: one in the control group and 2 in the treated group. One control foetus showed multiple anomalies: short forelimb, short hindlimb, flexed paw, dome shape of head and short body. Short hindlimb was also observed in one foetus of the treated group. All changes were considered incidental. Slight changes in the skeletal examination of the foetuses were noted between the treated and the control group and were considered spontaneous in origin and not related to treatment. Visceral examination of foetuses showed unilateral cryptorchidism (ectopic testis positioned just below the kidney) in 2 foetuses from 2 different litters (Dam numbers 55 and 75) of the treated group. The identification of the same stock male being mated with these two females (male no. 4) gave rise to the hypothesis that the male parent could genetically transmit the finding. In addition, considering also the high presence of displaced testes in the control group, the findings described were considered evidence of spontaneous pathology, often seen in this species under our experimental conditions.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1. Body weight of pregnant females (g) as group mean data.

Group	Day of gestation phase
		0	6	9	12	15	20
Control	n	24	24	24	24	24	24
	Mean	243.06	272.67	285.87	302.99	324.65	408.35
	SD	14.42	13.62	13.24	14.33	14.67	22.33
Treatment group	n	23	23	23	23	23	23
	Mean	240.36	269.66	282.13	299.15	322.08	408.67
	SD	13.94	12.77	12.47	14.1	16.75	22.36

Table 2. Litter data and sex ratios as group mean data.

Group		Corpora Lutea	Implantations	Uterine Deaths			Viable Young. Total	% Males	Implantation loss			Litter weight (g)	Mean foetal weight (g)
				Early	Late	Total			Pre	Post	Total
Control	n	24	24	24	24	24	24	24	24	24	24	24	24
	Mean	16.58	15.0	0.42	0.21	0.63	14.88	52.93	7.01	3.80	10.36	55.57	3.75
	SD	2.38	2.72	0.65	0.83	0.97	2.61	13.05	8.82	5.84	9.69	10.40	0.37
Treatment group	n	23	23	23	23	23	23	23	23	23	23	23	23
	Mean	17.83	17.17	0.52	0.52	1.04	16.13	50.75	3.77	5.99	9.45	57.97	3.59*
	SD	1.59	2.04	0.79	2.09	2.12	2.78	8.02	5.90	12.33	13.40	10.33	0.19

* = mean value of group is significantly different from controls at p < 0.05 (William’s test)

Table 3. Litter results.

	Control	Treatment group
Anomalies
Small foetus	1	2
Short forelimb, flexed paw, dome shape of head and short body	1	0
Short hindlimb	1	1
Visceral examination
Unilateral cryptorchidism	0	2

Conclusions:

The test substance had no effect on intrauterine development.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05 Jul 1994 - 15 May 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Administration of test article only during organogenesis.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 1981

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 1992

Deviations:

yes

Remarks:

treatment only during organogenesis (6-15) period

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles Rover, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 198 g
- Housing: animals were housed individually in Makrolon M3 cages with standard softwood bedding.
- Diet: pelleted Altromin Maintenance Diet 1324, Lot No. 170994/1340 (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 47-82
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE PHASE: 05 Jul 1994 - 27 Jul 1994.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% sodium carboxymethylcellulose and 0.25% Cremophor in aqua dest.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test article was prepared daily before administration.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- They were received at the testing facility on Day 0 of gestation.

Duration of treatment / exposure:

Day 6-15 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

Day 20 of gestation

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were based on the results of toxicological examinations (no further information, Henkel Report TBD 710070).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (working days) for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily (working days) for signs of reaction to treatment and/or symptoms of illness

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0, 6, 16 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: Gross macroscopic examination of all maternal organs with emphasis on the uterus, uterine contents, and position of foetuses in the uterus.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-test, based on a pooled variance, was applied for the comparison between the treated groups and the control group. The Steel-test was applied when the data could not be assumed to follow a normal distribution. Fisher’s Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No mortalities occurred during the study period. No compound-related symptoms were observed in the treatment groups in comparison to the control group. Body weight, body weight gains and corrected body weight were within expected ranges. No compound related differences were noted between the mean reproduction data of the test groups in comparison to the control group. At scheduled necropsy no macroscopic changes were noted in the dams of the treatment groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 900 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No substance-related symptoms were observed in the treatment groups. Pre-implantation loss, post-implantation loss, mean number of resorptions, embryonic deaths and total foetuses were not affected by treatment. Only one dead foetus was observed in the middle- and one dead fetus in the high-dose group from 345 and 306 live foetuses, respectively. Mean foetal placental and uterus weights were not affected by treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related malformations. The figures of visceral variations in the test groups were considered to be similar to the control group.
The mean weight of live male and female foetuses in the mid-dose group was significantly increased. The weights of live foetuses of the other treatment groups exhibited no significant differences on a litter and individual basis e.g. mean weight in comparison to the control group.
The figures of skeletal ossifications and variations showed no treatment-related deviations. The statistically significant differences concerning increased or decreased figures of various findings were considered to be incidental. Furthermore, no dose-relationship in any finding was observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 900 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1. Results of study.

Parameter	Group 1 0 mg/kg bw	Group 2 100 mg/kg bw	Group 3 300 mg/kg bw	Group 4 900 mg/kg bw
Number of corpora lutea [total/number of dams ± SD]	16.7 ± 1.9	17.1 ± 2.0	16.7 ± 1.9	16.5 ± 1.4
Implantations [total/number of dams ± SD]	15.3 ± 2.2	15.4 ± 2.7	14.9 ± 2.3	14.8 ± 1.5
Pre-implantation loss	34	39	44	38
Post-implantation loss	15	18	13	20
Embryonic death [total]	15	18	12	19
Embryonic resorptions [total/number of dams ± SD]	0.6 ± 1.1	0.7 ± 1.1	0.5 ± 0.7	0.7 ± 0.9
Foetal resorptions [total/number of dams ± SD]	0.0 ± 0.2	0.0 ± 0.2	0.0 ± 0.0	0.1 ± 0.4
Live foetuses	336	337	345	306
Dead foetuses	0	0	1	1
Malformed foetuses [total/number of dams ± SD]	0.0 ± 0.2	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.2
Sex of foetuses (%males : % females)	47.3 : 52.7	51.6 : 48.4	50.3 : 49.4	52.1 : 47.6
Weights of live foetuses (mean ± SD) Males Females	4.3 ± 0.9 4.0 ± 0.8	4.2 ± 0.8 4.0 ± 0.7	5.0 ± 0.9* 4.6 ± 0.8*	4.5 ± 0.7 4.3 ± 0.7
Weights of placenta (mean ± SD)	0.6 ± 0.1	0.6 ± 0.1	0.6 ± 0.1	0.6 ± 0.1
Weights of uteri (mean ± SD)	88.7 ± 14.9	89.3 ± 19.2	97.9 ± 18.9	90.9 ± 11.7

*: Dunnett-test based on pooled variance, significant at level 5%

Table 2. Skeletal examination of foetuses (stage of development).

Parameter (%)	Group 1 0 mg/kg bw	Group 2 100 mg/kg bw	Group 3 300 mg/kg bw	Group 4 900 mg/kg bw
Foetal skeleton No abnormal findings	4.0	9.8	16.1##	10.0
Skull bones, single incompletely ossified	4.5	11.6#	10.0	5.0
Sternebrae incompletely ossified abnormally ossified	66.5 24.4	84.4## 17.9	51.1## 9.4##	66.3 17.5
Coccygeal vertebrae, 4 and more ossified	32.4	22.5	56.1##	47.5#
Pelvis, pubis: incompletely ossified	6.2	0.6#	0.6##	1.3

#/##: Fishers exact test (two-sided) significant at level 5% (#) or 1% (##), Bonferroni-Holm-corrected

Conclusions:

The test substance had no effect on intrauterine development.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Nov 2000 to 30 Nov 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

No guideline mentioned in study report

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 1998

Deviations:

yes

Remarks:

no immunological effects addressed

Principles of method if other than guideline:

No guideline mentioned in study report

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
- Source: Harlan ltaly S.r.l., 33049 San Pietro al Natisone (UD), Italy
- Females (if applicable) nulliparous and non-pregnant: not stated
- Age at study initiation: 27-29 days old
- Weight at study initiation: 89.0 - 102.1 g for males and 84.6 - 101.7 g for females
- Fasting period before study: none
- Housing: 5 of one sex to a cage, clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Code 1354 G, Techniplast Gazzada S.a.r.1., Buguggiate, Varese). Each cage tray held absorbent paper which was inspected daily and changed three times a week.
- Diet: Altromin MT pelleted diet, A. Rieper, Bolzano, ltaly, ad libitum
- Water: via water bottles, ad libitum
There was no information indicating that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or in the diet.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 16 Nov 2000 to 22 Feb 2001

Route of administration:

oral: gavage

Details on route of administration:

Dose volume of 2 mL/kg body weight

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was dissolved in the vehicle (corn oil). The formulations were prepared daily. The dose was administered to each animal on the basis of the most recently recorded body weight, and the volume administered was recorded for each animal.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not given
- Concentration in vehicle: 125, 250 and 500 mg/mL
- Lot/batch no. (if required): not given
- Purity: not given

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

None

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not given

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: For mortality twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre and post-dose observations of individual animals for signs of reaction to treatment was carried out daily before dosing, approximately 30 minutes and 2 hours after dosing. Once before commencement of treatment and once a week thereafter each animal was subjected to a detailed clinical examination, which included an evaluation of neurotoxicity. Animals were examined in an open arena for a period of three minutes: removal (from cage), handling reactivity, lacrymation, palpebral closure, salivation, piloerection, rearing, motility impairment, arousal (animal activity), vocalisation, stereotypies, unusual respiratory pattern, bizarre behaviour, urination, defecation, clonic and tonic movements, and gait.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
The weight of food consumed by each cage of rats was recorded weekly following allocation and the group mean daily intake per rat was calculated.

FOOD 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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals assigned to the study were examined just prior to the start of treatment, by means of both an ophthalmoscope and a slit-lamp microscope, after the instillation of 0.5% tropicamide (Visumidriatic, Merck, Sharp and Dohme, Rome, ltaly). The eyes of all animals in all groups were re-examined during week
13 of treatment. During the examination particular attention was paid to: anterior chamber, conjunctivae and eyelids, cornea and sclera, iris, lens, posterior segment - vitreous humour, and ocular fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 13 samples of blood were withdrawn under light ether anaesthesia from the retro-orbital sinus of all rats from each group after overnight fasting.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: haematocrit, haemoglobin, red blood cell count, reticulocyte count (not carried out as there were no signs of anaemia), mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), abnormalities of the blood film, platelets, and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 13 samples of blood were withdrawn under light ether anaesthesia from the retro-orbital sinus of all rats after overnight fasting.
- Animals fasted: Yes
- How many animals: all
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, albumin, total bilirubin, total cholesterol, total protein, sodium, phosphorus, potassium, calcium, and chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: During week 13 samples of overnight urine samples were collected from all rats
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, collection of urine during overnight fasting
- Parameters checked: appearance, volume, specific gravity, pH, protein, total reducing substances, glucose, ketones, bilirubin, urobilinogen, blood, and sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the study
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
The Motor Activity of the first 5 males and 5 females was measured once during week 12 of treatment by an automated activity recording device (Mini Opto-V arimex, Columbus International Corp.). Measurements were performed using a computer generated random order.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, broad range of organs

Organ weights: adrenal glands, brain, epididymides, heart, liver, kidneys, ovaries, spleen, testes, thymus, and uterus.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Microscopic observations were tested for statistical significance using the non-parametric Kolmogorov-Smirnov test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation, mainly slight in severity, was observed in some treated as well as control animals during the study. The incidence of this sign was higher in treated males than in controls while the incidence in treated female groups was almost comparable to control animals. As this sign was noted in both control and treated animals it could be considered to be due to the vehicle used for the administration of the test item.
At the daily pre- and post-dose observations, salivation was occasionally noted just before dosing or 30 minutes after dosing, in a few male and female animals from the low, mid- and high-dose groups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No changes in body weight were observed in males and females from mid- and high-dose groups and females from the low-dose group. However, a reduced body weight increment was observed in low-dose males when compared to controls, mainly from the 3rd to the 6th week of dosing, causing a change in body weight, which achieved statistically significance in most ofthe measurements performed from the 4th week to the end of treatment.
This change, noted in only one sex receiving the low dose-level, was not considered treatment-related.
Terminal body weight was significantly reduced in low-dose males.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Reductions in food consumption of low-dose males were observed on 4 occasions during the study (weeks 2, 3, 5 and 10).
Low-dose females and mid- and high-dose animals did not show any changes in food consumption when compared to controls throughout the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant macroscopic changes were detected in the animals killed at termination which could be considered related to the test item. The macroscopic observations reported did not reveal any significant difference between treatment and control animals killed at termination.
Statistically significant reductions in absolute liver and kidney weights were achieved in low and high-dose males andin relative weights in mid and high-dose males while the absolute liver weight was increased in low-dose females. In addition, absolute spleen weight was increased in mid- and high-dose females and absolute and relative uterus weight was decreased in low-dose females. All these changes were considered to be of no biological relevance as they were not consistent between sexes and not related to any morphological changes at the microscopic examination.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histopathological changes detected did not show any evident differences between the treated and control animals that could be considered treatment-related.
The seminiferous tubules in the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell type present within the different stages. The findings observed were considered to be an expression of spontaneous pathology normally seen in this species.

Histopathological findings: neoplastic:

no effects observed

Details on results:

No abnormalities were detected at the macroscopic observation and histopathological examination of the organs of the reproductive systems such as prostate gland, seminal vesicles, testes and epididymides in males and ovaries and uterus in females. The weights of testes, epididymides, ovaries and uterus were not affected by treatment. In addition, following the evaluation of the seminiferous tubules with respect to their stage in the spermatogenic cycle and the integrity of the various cell type present within the different stages, no abnormalities were noted.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

highest dose tested

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Key result

Critical effects observed:

no