Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A well conducted 2-generation study was available in rats.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 June 2008 to 09 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Rat: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) 5-6 wks
- Weight at study initiation: (P) Males: 147-182 g.; Femaels: 126-150 g.
- Fasting period before study: no
- Housing:
Pre-mating Animals were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 4 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation Offspring was kept with the dam until termination.
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): Free access to prepared diets. During the acclimatisation period, animals had free access to the same diet (without the test substance) received from the supplier in pelleted form (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2-23.4°C
- Humidity (%): 21-92%.
Temporary deviations from the maximum and minimum level of relative humidity occurred. Based on laboratory historical data this was not considered to have influenced the study outcome.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

IN-LIFE DATES: From: 30 June 2008 To: 09 March 2009
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed (without the use of a vehicle) directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.
On one occasion the pellets were dried for approximately 44 hours instead of approximately 24 hours. Analytical results were in the same range as diets dried for 24 hours. Therefore, an extension of the drying period for 20 hours is not considered to have affected the quality of the pellets.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared at least once every 22 days.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept at room temperature in the diet store room in the animal house.
Details on mating procedure:
Following a minimum of 70 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates for the F0-generation and for the F1-generation parentage was known through mating records maintained in the raw data). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 15 days was allowed for mating. If no evidence of copulation was obtained after 10 days, the female was placed with another male (for an additional five days) of the same treatment group who had successfully mated, again avoiding sibling mating.

- M/F ratio per cage: 1/1
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were done according to a validated method (NOTOX Project 488600) in Week 11, 22, 33

In addition, random samples from all diet preparations not yet selected for analytics (Weeks 3, 5, 7, 9, 13, 15, 17, 23, 25, 27, 29, 31, 35) were taken and analyzed for accuracy (Groups 1-4). These random samples from all diet preparations that were stored at ≤-15°C for possible future analysis and an additional sample of 200-300 gram of Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) were dispatched to Wiljam Nijenhuis, Akzo Nobel Chemicals bv, Research, Development & Innovation, Building R5, Zutphenseweg 10, P.O. Box 10, 7400 AA Deventer, The Netherlands. Additional verification of dose concentrations were performed by the sponsor under non-GLP conditions. These results were added as an appendix to the study report.
Duration of treatment / exposure:
The F0-generation was exposed for a minimum of 70 days prior to mating and continuing until euthanasia. The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 70 days prior to mating and continuing until euthanasia. The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.
Frequency of treatment:
Ad libitum
Details on study schedule:
- F1 parental animals not mated until 70 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 15-16 weeks (P) and 13 weeks (F1)
Remarks:
Doses / Concentrations:
0, 625, 2500 and 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
24 per sex per dose with exception of group 4 (F1 generation):
In total 23 female F1-pups were selected for Group 4 instead of 24
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of the 14-day dose range finding study (NOTOX Project 488325).
- Rationale for animal assignment (if not random): random
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
At least once daily, detailed clinical observations were made in all animals. The time of onset, degree and duration was recorded.
On three days during the study period, no (on-line) recording of clinical signs was performed. Clinical signs data of these days are therefore missing in the tables in Appendix 1 to 4. Sufficient data from other days was available for evaluation.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 of post-coitum and during lactation on Days 1, 4, 7, 14 and 21.
For one animal (F0 generation, Group 1), erroneously no body weights and food consumption were measured on Day 0 post-coitum. On Day 1 of the mating period no body weights were recorded for parenteral F1 males and females. As a result no body weight gain results are available for the mating period of these animals. Sufficient data is available for evaluation of effects on body weight.

To better monitor one animal and its pups (F0 generation, Group 4), additional measurements (body weight, water and/or food consumption) were done on this animal on 8 days during the study period (16 to 23 October 2009, last days before necropsy). Results were recorded in the raw data. The results were used for the evaluation of the condition of the animal only.

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
Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1, 4, 7, 14 and 21.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. The actual test substance intake for each period was estimated based on the body weight and food consumption values.

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect on water intake was suspected.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 21 days prior to initiation of the mating period and until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also prepared to determine the stage of estrous.

The stages include e (=estrus), d (=di-estrus), m (=met-estrus), p (=pro-estrus), u (= unable to determine estrous stage/length of estrous cycle) and s (= smeared positive for sperm).

An analysis of the female cycle pattern was reported. The cycle patterns were classified as:
R = Regular (all cycles either 4 or 5 days);
IR = Irregular (at least one cycle of 2, 3 or 6-10 days, irrespective of the number of 4-5 day cycles);
A = Acyclic (least 10 days without estrus, beginning prior to pairing irrespective of the number of 4-5 day cycles);
EE = Extended Estrus (at least 4 consecutive days of estrus);
ED = Extended Di-Estrus during pairing (at least 5 consecutive days of di-estrus during pairing).

Cycle classifications for individual animals were based on the length and stage of each estrous cycle (beginning with the first day of dose administration until evidence of mating was detected).
Sperm parameters (parental animals):
Parameters examined in [F0- and F1] male parental generations:
For all surviving males, the following assessments were performed:
From all males, sperm samples were taken from the proximal part of the vas deferens (right):
1. Sperm motility was assessed of all samples.
2. Sperm smears for morphological evaluation were fixed of all samples. Abnormal forms of sperm from a differential count of 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed for ten randomly selected samples of males of the control and high dose group.
One testis and one epididymis (left) from all males were removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left testis and epididymis were weighed, homogenized and evaluated for sperm numbers. The sperm production rate was calculated utilizing the method described by Blazak et al. (Blazak, 1985). Evaluation was performed for all samples of the control and high dose group.

In summary:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

To reduce variability among the litters, on Day 4 of lactation, eight pups from each litter of equal sex distribution (if possible) were randomly selected. For litters consisting of fewer than eight pups, adjustments for litter size was not performed. In the litter of one female (F0 generation, Group 2), one pup was culled on Day 7 of lactation instead of Day 4. A three days delay in culling of one pup has no effect on the data.

PARAMETERS EXAMINED
The following parameters were examined in [F1 and F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities:
- Mortality / Viability: The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights Live pups were weighed during lactation on Days 1, 4, 7 and weekly thereafter.
- Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).
- Balanopreputial separation: Each selected male pup (24 rats/group) was observed for balanopreputial separation (prepuce opening) beginning on postnatal Day 35 (Korenbrot, 1977). Examination of the males continued daily until balanopreputial separation was present. The body weight of each male was recorded on the day of acquisition of balanopreputial separation.
- Vaginal perforation: Each selected female pup (24 rats/group) was observed for vaginal perforation (vaginal opening) beginning on postnatal Day 25 (Adams, 1985). Examination of the females continued daily until vaginal perforation was present. The body weight of each female was recorded on the day of acquisition of vaginal perforation.
- Anogenital distance: Due to a slight delay in balanopreputial separation and vaginal opening for F1 pups in the high dose group, anogenital distance was measured for all live F2 pups on Day 1 of lactation.

In the litter of one female (F0 generation, Group 2), one pup was overlooked on Day 1. This pup was discovered on Day 5 of lactation. On Day 5 of lactation, the body weight was recorded (8.01 gram) in the raw data and subsequently the pup was culled. No data for this animal was recorded in the tables. For the pups of one female (F0 generation, Group 3) no body weights were recorded from Days 1 to 14 of lactation as the starting date of the pups (Day 1 of lactation) was not recorded correctly. Sufficient information was available from other litters for evaluation.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving F0-and F1-males were killed as soon as possible after delivery of the litters.
- Maternal animals: All surviving F0-and F1-females were killed on Day 21 post partum or shortly thereafter.

GROSS NECROPSY
Gross necropsy consisted of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Of all paired females, the number of former implantation sites in the uterus was assessed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated ibelow were prepared for microscopic examination and weighed, respectively:
Identification marks: not processed
(Pituitary gland)
Adrenal glands
Prostate gland
(Brain (cerebellum, mid-brain, cortex))
Seminal vesicles
Cervix
Spleen
Coagulation gland
Testes
Epididymides
Thyroid including parathyroid (if detectable)
Kidneys
Uterus
Liver
Vagina
Ovaries
All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

A few tissues/organs were not available for histopathology and organ weights. These tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Tissues are listed in the raw data and pathology report.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
Pups, younger than 7 days, were killed by decapitation. All remaining pups were sacrificed using an oxygen/carbon dioxide procedure.
Culling was performed on Day 4 of lactation or shortly thereafter. The remaining pups (excluding F1-pups selected for mating) were killed at Day 21 post partum or shortly thereafter.
- Animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Stillborn pups and pups dying between birth and Day 4 of lactation were sexed and dissected (including the heart and the brain examined by a mid-coronal slice) by a technique described by Stuckhardt and Poppe (Stuckhardt, 1984). These examinations were only performed when practically possible (e.g. in the absence of cannibalism, autolysis). Culled offspring was sexed and externally examined with emphasis on developmental morphology. The stomach was examined for the presence of milk. All nonselected F1 and F2 weanlings were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded. All gross lesions were collected and placed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

GROSS NECROPSY
- Gross necropsy consisted of thoracic and abdominal tissues and organs with emphasis on developmental morphology.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
Brain
Liver: not weighed
Spleen
Thymus
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 (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
- The T-test (Gossett, 1908) was applied to sperm concentrations in the testis and epididymis.
- The percentage of motile spermatozoa, progressive motile spermatozoa and sperm with normal morpholpgy were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data to compare the treated groups to the control group.

All tests were two-sided and in all cases p<0.05 was accepted as the level of statistical significance.
Reproductive indices:
Percentage mating males: Number of males mated/Number of males paired x 100
Percentage mating females: Number of females mated/Number of females paired x 100
Fertility index males: Number of males generating a pregnancy/Number of males paired x 100
Fertility index females: Number of pregnant females/Number of females paired x 100
Conception rate: Number of pregnant females/Number of females mated x 100
Gestation index: Number of females bearing live pups/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Offspring viability indices:
Percentage of postnatal loss Days 0-4 post partum: Number of dead pups on Day 4 post partum/Number of live pups at First Litter Check x 100
Percentage of breeding loss Day 5 until weaning: Number of dead pups between Days 5 and 21 post partum/Number of live pups on Day 4 post partum x 100
Percentage live males at weaning: Number of live male pups on Day 21 post partum/Number of live pups on Day 21 post partum x 100
Percentage live females at weaning: Number of live female pups on Day 21 post partum/Number of live pups on Day 21 post partum x 100
Viability index: Number of live pups on Day 4 post partum/Number of pups born alive x 100
Weaning index: Number of live pups on Day 21 post partum/Number of live pups on Day 4 post partum x 100
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
OBSERVATIONS F0-GENERATION
F0-GENERATION Mortality:
No mortality occurred.

F0-GENERATION Clinical signs:
No treatment related clinical signs were observed.

Incidental findings consisted of alopecia and/or scabs on different parts of the body, chromodacryorrhoea, scales (inguinal region) and a bleeding nose. These findings are occasionally seen among rats used in this type of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

F0-GENERATION Body weights:
Decreased body weights and body weight gain were observed for animals treated at 10000 ppm. Body weights were statistically significantly decreased for almost the complete treatment period and body weight gain was statistically significantly lower during the premating period and during a few days during post-coitum (Days 17-20) and lactation (Day 4).

No treatment related effect on body weights was noted for animals treated at 625 and 2500 ppm. A slight increase in body weight gain was noted in females treated at 2500 ppm on Day 57 of the premating period. This finding was considered to be of no toxicological relevance in view of the single occurrence and because in case of a toxic response, a decrease rather than an increase in body weight gain would be expected.

F0-GENERATION Food consumption:
Decreased absolute food consumption was observed in animals treated at 10000 ppm (not always statistically significant) during almost the complete treatment period. After allowance for body weight, a slight decrease in food consumption was only noted during lactation (not always statistically significant). No treatment related effect on food consumption was noted for animals treated at 625 and 2500 ppm.

All other statistically significant findings were minor and considered to be of no toxicological relevance as these changes were slight, a dose response relationship was lacking, and/or these changes were within the normal range of variation for rats of this age and strain.

F0-GENERATION Test article intake:
The mean test article intake values (mg substance/kg body weight/day) are summarized in attached tables:
Mean test article intake based on nominal dose level of the diet; see attachment: Table 1.
Mean test article intake when corrected for mean value of recovery; see attachment: Table 2.

F0-GENERATION Macroscopic examinations:
The following macroscopic abnormalities were seen:
- Black-brown discolouration of the liver in all males and females at 10000 ppm, and in 1/24 females at 2500 ppm,
- Enlarged liver in 2/24 males and 8/24 females at 10000 ppm,
- Greenish to black-brown discolouration of the kidneys in 21/24 males and all females at 10000 ppm, and in 1/24 males and 1/24 females at 2500 ppm,
- Reduced size of the adrenal glands in 11/24 males and 4/24 females at 10000 ppm,
- Pale to black-brown discolouration of the adrenal glands in 20/24 males and 15/24 females at 10000 ppm, in 3/24 males and 3/24 females at 2500 ppm and in 1/24 males and 1/24 females at 625 ppm,
- Enlarged renal lymph nodes in 12/24 males and 16/24 females at 10000 ppm and in 2/24 males at 2500 ppm,
- Black-brown to black discolouration of the renal lymph nodes in 17/24 males and 22/24 females at 10000 ppm and in 7/24 males at 2500 ppm,
- Enlarged hepatic lymph nodes in 3/24 females at 10000 ppm,
- Black discolouration of the hepatic lymph nodes in 1/24 males and 2/24 females at 10000 ppm,
- Enlarged spleen in 2 males at 10000 ppm.
These findings were considered to have been caused by the treatment.

Watery fluid in the uterus, found in three females of the control, low and mid dose group is related to a stage in the oestrous cycle and is a normal finding.

Enlarged and discolouration of the renal lymph node was also noted in one animal at 625 ppm. However, in absence of a dose response relationship and as this finding was noted in one animal only, this effect was not considered to represent a toxicologically relevant finding. Incidental findings included alopecia, diaphragmatic hernia, pelvic dilation of the kidneys, foci on the liver or thymus, enlarged kidneys, reduced size of seminal vesicles, enlarged or reduced size of the thyroid, discoloured spleen or thymus and a cyst on the kidney. These findings are occasionally seen among rats used in these types of studies. In addition, because of the low incidence observed, or in the absence of a treatment-related distribution they were considered changes of no toxicological significance.

F0-GENERATION Organ weights:
The following statistically significant changes in organ weights distinguished treated groups from the control group:
- Increased absolute liver weights and liver to body weight ratios in males at 2500 and 10000 ppm and in females at 10000 ppm,
- Increased kidney to body weight ratio in males and females at 10000 ppm,
- Decreased absolute adrenal weight and adrenal to body weight ratio in females at 10000 ppm,
- Increased absolute spleen weight in males at 10000 ppm and increased spleen to body weight ratio in males and females at 10000 ppm,
- Increased testes to body weight ratio in males at 10000 ppm,
- Increased epididymides to body weight ratio in males at 10000 ppm,
- Decreased absolute ovaries weight and ovaries to body weight ratio in females at 10000 ppm,
- Decreased absolute uterus weight and uterus to body weight ratio in females at 10000 ppm.

The following changes were considered to be related to the decreased body weights in treated high dose animal:
- Increased brain to body weight ratio in males and females at 10000 ppm,
- Slightly decreased absolute adrenal weight in males at 10000 ppm
- Slightly decreased absolute pituitary weight in females at 10000 ppm,
- Slightly decreased absolute thyroid weight in females at 10000 ppm,
- Slightly decreased absolute prostate weight in males at 10000 ppm,

All other statistically significant findings were considered to be of no toxicological relevance in absence of a dose response relationship.

F0-GENERATION Microscopic examination:
The following microscopic findings were noted:
- Biliary/peribiliary brown pigment in the liver of 22/24 males and 24/24 females (minimal to marked) at 10000 ppm and in 7/10 males (minimal to moderate) and 1/10 females (minimal) at 2500 ppm,
- Peribiliary inflammation in 18/24 males (minimal to moderate) and 22/24 females (minimal to slight) at 10000 ppm, in 9/10 males and 8/10 females (minimal to slight) at 2500 ppm. Minimal peribiliary inflammation was also observed in 4/11 males and 4/10 females at 625 ppm and 3/10 males and 1/9 females of the control group,
- Centrilobular hepatocellular hypertrophy in 21/24 males and 21/24 females (minimal to moderate) at 10000 ppm,
- Bile duct proliferation in 23/24 males and 23/24 females (minimal to marked) at 10000 ppm and in 9/10 males (minimal to moderate) and 1/10 female (minimal) at 2500 ppm,
- Tubular pigment in the kidneys of 21/21 males (minimal to slight) and 24/24 females (minimal to moderate) at 10000 ppm. Tubular pigment at a minimal degree was also observed in 10/11 males and 5/10 females at 2500 ppm, in 5/10 males and 1/10 females at 625 ppm and in 5/10 males and 2/10 females of the control group,
- Lymphoid hyperplasia in the renal lymph nodes of 17/17 males (slight to marked) and 21/22 females (minimal to marked) at 10000 ppm and of 7/7 males (minimal to moderate) at 2500 ppm,
- Macrophage infiltration in the renal lymph nodes of 16/17 males and 22/22 females (minimal to moderate) at 10000 ppm and of 6/7 males (minimal to slight) at 2500 ppm,
- Brown pigment in the renal lymph nodes of 17/17 males (slight to marked) and 22/22 females (minimal to marked) at 10000 ppm and of 7/7 males (slight to moderate) at 2500 ppm,
- Lymphoid hyperplasia in the spleen of 7/11 males and 6/10 females (minimal to slight) at 10000 ppm. Minimal lymphoid hyperplasia was also observed in 3/10 females at 2500 ppm, in 2/10 males at 625 ppm and in 3/10 males and 3/10 females of the control group,
- Lymphoid hyperplasia, brown pigments and/or macrophage infiltration of the hepatic and/or pancreatic lymph nodes in individual animals at 10000 ppm.

The special stains performed on liver sections of 6 animals were all negative, i.e. the observed biliary/peribiliairy brown pigment contained neither iron nor bile pigments.

Quantitative assessment of primordial follicles in the ovaries of F0 females showed no difference between control and high dose animals.

In the F0-generation, nineteen animals were recorded as suspected infertile (11 males and 8 females). These were spread among the dose groups with no obvious relationship to treatment. In two males (one at 2500 ppm and one at 10000 ppm) minimal or slightseminiferous atrophy was noted, but this was not considered to be related to treatment. No other morphologic causes for infertility could be determined.

All other microscopic findings were within the range of background pathology encountered in Wistar Han rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

F0-GENERATION Sperm motility, concentration and morphology:
No effect on sperm motility, concentration and morphology was noted up to 10000 ppm.

F0-GENERATION Estrous cycle:
The percentage of females per group that were classified as having a ‘regular’ cycle were 100.0, 100.0, 91.7, and 75.0% at 0, 625, 2500 and 10000 ppm, respectively. In these animals, the percentage of irregularities was 0%, 0%, 8.3% and 25%, respectively. Furthermore, at 10000 ppm, the duration of the estrous cycle varied between 3 and 12 days, compared to 4 days for all animals in the control group.

F0-GENERATION Reproduction:
Reproduction parameters were unaffected by treatment up to 10000 ppm.

All fertility parameters were well within the normal range of variation in rats of this age and strain. The mean number of implantation sites (as described in Appendix 2) at the high dose group was slightly lower compared to the control group. However, the difference was not statistically significant and the values were within normal limits.

For reproduction Data F0-generation; see attachment; Table 3.
For fertility F0-generation; see attachment; Table 4.

F0-GENERATION Breeding data:
The mean number of living pups at first litter check was statistically significantly lower in animals treated at 10000 ppm (10.0 pups per litter at 10000 ppm when compared to 11.8 pups in the control group).

No other treatment-related effects were noted. Although there was no effect on the number of litters showing postnatal loss, total postnatal loss was statistically significantly increased in animals at 10000 ppm which was mainly due to one female that had 8 missing pups on Day 4 of lactation. Before Day 4 of lactation no clinical signs were noted in these pups, only a slight decrease in body weights when compared to concurrent controls. As a result of this effect on postnatal loss also the viability index was slightly lower, but remained within the normal range of variation. Breeding parameters were unaffected by treatment at 625 and 2500 ppm.


OBSERVATIONS F1-GENERATION (PARENTAL ANIMALS)
F1-GENERATION Mortality:
No mortality occurred.

F1-GENERATION Clinical signs:
No treatment-related clinical signs were observed.

Piloerection was noted in one female at 2500 ppm and two males and two females at 10000 ppm for a maximum of 10 days. Because of the low incidence observed these findings were considered to be of no toxicological relevance.

Incidental findings consisted of alopecia and/or scabs on different parts of the body, broken tail (apex), hunched posture, lean appearance, swelling of the toes, scales and purulent discharge from the eye. These findings are incidentally seen among rats used in these types of studies. At the incidence observed or in the absence of a treatment-related distribution these were considered changes of no toxicological significance.

F1-GENERATION Body weights:
Decreased body weights were observed for animals treated at 10000 ppm during the complete treatment period with a slight decrease in body weight gain on Day 20 post-coitum. Slight decreases in body weights were also noted at 2500 ppm during lactation (not always statistically significant). No treatment related effect on body weights were noted for animals treated at 625 ppm.

Changes in body weight gain at 625 ppm were considered to be of no toxicological relevance as body weights were already high on Day 1 of premating. Also no relevance was attached to the slight change in body weight gain at 2500 ppm on Day 7 of lactation in the absence of a dose response relationship.

F1-GENERATION Food consumption:
Decreased absolute food consumption was observed for animals treated at 10000 ppm (not always statistically significant) during almost the complete treatment period. However, after allowance for body weight, a slight increase in food consumption was noted at 10000 ppm (not always statistically significant).

Incidentally, slight increases in relative food consumption were also noted at 625 and 2500 ppm. Because of the low incidence observed, and as values were within the normal range of variation, these changes were considered to be of no toxicological relevance. All other statistically significant findings were minor and considered to be of no toxicological relevance in the absence of a dose response relationship.

F1-GENERATION Test article intake:
The mean test article intake values (mg substance/kg body weight/day) are summarized in attached tables:
Mean test article intake based on nominal dose level of the diet; see attachment: Table 5.
Mean test article intake when corrected for mean value of recovery; see attachment: Table 6.

F1-GENERATION Macroscopic findings:
The following macroscopic abnormalities were considered to be related to treatment with the test substance:
- Black-brown discolouration of the liver in 23/24 males and all females at 10000 ppm, and in 1/24 males and 4/24 females at 2500 ppm,
- Black-brown discolouration of the kidney in 4/24 males and 14/23 females at 10000 ppm, and in 1/24 males at 2500 ppm,
- Pale discolouration of the adrenal glands in 15/24 males and 3/23 females at 10000 ppm, and in 7/24 males at 2500 ppm,
- Reduced size of the adrenal glands in 4/23 females at 10000 ppm,
- Enlarged renal lymph nodes in 23/24 males and all females at 10000 ppm and in 24/24 males and 22/24 females at 2500 ppm,
- Black-brown to black discolouration of the renal lymph nodes in 23/24 males and all females at 10000 ppm and in 24/24 males and 22/24 females at 2500 ppm,
- Enlarged hepatic lymph nodes in 2/24 males at 10000 ppm,
- Black-brown/red-brown discolouration of the hepatic lymph nodes in 2/24 males and 2/23 females at 10000 ppm,
- Black-brown discolouration of the pancreatic lymph nodes in 1/23 females at 10000 ppm and in 1/24 females at 2500 ppm.

Watery fluid in the uterus, found in five control females, eight low dose females, two mid dose females and one high dose female is related to a stage in the oestrous cycle and is a normal finding.

Incidental findings included alopecia, tail bent, foci on the urinary bladder, thymus and diaphragm, reduced size of testes, seminal vesicles, epididymides and prostate, nodule in the pancreatic adipose tissue and diaphragmatic hernia of the liver. These findings are occasionally seen among rats used in these types of studies. Because of the low incidence or in the absence of a treatment-related distribution they were considered changes of no toxicological significance.

F1-GENERATION Organ weights:
The following statistically significant changes in organ weights distinguished treated from control animals:
- Increased pituitary to body weight ratio in females at 10000 ppm,
- Increased absolute liver weights and liver to body weight ratios in males and females at 2500 and 10000 ppm (absolute weight not statistically significant in females at 2500 ppm),
- Increased relative kidney weight to body weight ratio in males and females at 10000 ppm.
- Increased absolute spleen weight in males at 10000 ppm, and increased spleen to body weight ratio in females at 10000 ppm, and in males at 2500 ppm and 10000 ppm,
- Decreased absolute testes weights and increased testes to body weight ratio in males at 10000 ppm,
- Decreased absolute epididymides weights and increased epididymides to body weight ratio in males at 10000 ppm,
- Increased seminal vesicles to body weight ratio in males at 10000 ppm,
- Decreased absolute uterus weights and uterus to body weight ratios in females at 2500 and 10000 ppm.

The following changes were considered to be related to the decreased body weights in high dose animals:
- Decreased absolute brain weight and increased brain to body weight ratio in males and females at 10000 ppm,
- Slightly decreased absolute pituitary weight in males at 10000 ppm,
- Slightly decreased absolute thyroid weight in males at 10000 ppm,
- Slightly decreased absolute adrenal weight in males and females at 10000 ppm,
- Slightly decreased absolute prostate weight in males at 10000 ppm,
- Slightly decreased absolute ovaries weight in females at 10000 ppm.

F1-GENERATION Microscopic examination:
The following microscopic findings were considered to be related to treatment with the test substance:
- Biliary/peribiliary brown pigment in the liver of 24/24 males (slight to marked) and 23/23 females (moderate to marked) at 10000 ppm and of 11/11 males and 12/12 females (slight to moderate) at 2500 ppm and of 2/10 males (minimal) and 3/10 females (minimal) at 625 ppm.
- Peribiliary inflammation in 18/24 males and 19/23 females (minimal to slight) at 10000 ppm, in 11/11 males (minimal to moderate) and 11/12 females (minimal to slight) at 2500 ppm. Minimal peribiliary inflammation was also observed in 5/10 males and 4/10 females (minimal) at 625 ppm and in 2/10 males of the control group,
- Centrilobular hepatocellular hypertrophy in 19/24 males (minimal to slight) and 12/23 females (minimal to moderate) at 10000 ppm, 1/12 females (minimal) at 2500 ppm and in 1/10 females (minimal) at 625 ppm,
- Bile duct proliferation in 24/24 males and 23/23 females (slight to marked) at 10000 ppm and 11/11 males and 11/12 females (minimal to moderate) at 2500 ppm and in 2/10 females (minimal) at 625 ppm,
- Tubular pigment in the kidneys of 8/11 males and 16/16 females (minimal to moderate) at 10000 ppm. Tubular pigment at a minimal degree was also observed in 2/11 males and 4/10 females at 2500 ppm and in 5/10 females at 625 ppm,
- Lymphoid hyperplasia in the renal lymph nodes of 23/23 males (minimal to marked) and 23/23 females (slight to marked) at 10000 ppm and of 24/24 males and 22/22 females (minimal to marked) at 2500 ppm.
- Macrophage infiltration in the renal lymph nodes of 23/23 males (minimal to moderate) and 23/23 females (slight to marked) at 10000 ppm and of 24/24 males (minimal to moderate) and 22/22 females (minimal to marked) at 2500 ppm.
- Brown pigment in the renal lymph nodes of 23/23 males (minimal to moderate) and 23/23 females (slight to moderate) at 10000 ppm and of 24/24 males and 22/22 females (slight to marked) at 2500 ppm,
- Lymphoid hyperplasia in the spleen was observed in 4/10 males (minimal) and 4/10 females (minimal to slight) at 10000 ppm. Minimal lymphoid hyperplasia was also seen in 4/10 males and 2/10 females at 2500 ppm and in 1/10 females at 625 ppm,
- Lymphoid hyperplasia, brown pigments and/or macrophage infiltration of the hepatic and/or pancreatic lymph nodes in individual animals at 10000 ppm.

A total of twenty two animals in the F1 generation was recorded as suspected infertile (13 males and 9 females). These were spread across the dose groups with no relationship to treatment. In one male (at 2500 ppm) massive seminiferous atrophy in the testis and oligospermia in the epididymides were noted. No other morphologic causes for infertility could be determined.

All other microscopic findings were within the range of background pathology encountered in Wistar Han rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

F1-GENERATION Sperm motility, concentration and morphology:
No effect on sperm motility, concentration and morphology was noted up to 10000 ppm.

F1-GENERATION Estrous cycle:
The regularity and duration of estrus cycle were not affected by treatment.

The percentage of females per group that were classified as having a ‘regular’ cycle were 87.5, 75.0, 91.7, and 82.6% at 0, 625, 2500 and 10000 ppm, respectively. The percentage of irregularities was 13%, 26%, 8% and 17% at 0, 625, 2500 and 10000 ppm, respectively.

F1-GENERATION Reproduction:
Reproduction parameters were unaffected by treatment up to 10000 ppm. There was a trend towards a slightly decreased number of pregnant females and a slightly decreased conception rate and fertility index for females at 10000 ppm; however this was within the normal range of variation in rats of this age and strain.

For reproduction Data F1-generation; see attachment: Table 7.
For fertility F1-generation; see attachment: Table 8.

F1-GENERATION Breeding data:
The mean number of living pups at first litter check was slightly lower in animals treated at 10000 ppm (10.3 pups per litter at 10000 ppm when compared to 11.7 pups in the control group). The total number of living pups was also decreased, which was also partly due to the slightly reduced number of pregnant females. No increase in postnatal loss, breeding loss, weaning index or viability index was noted. Breeding parameters were unaffected by treatment at 625 and 2500 ppm.
Dose descriptor:
NOAEL
Effect level:
625 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects on body weight; food consumption; organ weights; histopathology. 625 ppm corresponded to 29-36 mg/kg bw/day
Dose descriptor:
other: Reproduction NOAEL
Effect level:
> 10 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproduction parameters were unaffected up to 10000 ppm; 10000 ppm corresponded to 488-591 mg/kg bw/day
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
only in F2 pups
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
only in F2-pups, not examined in F1-pups
F1-PUP DEVELOPMENT
F1-PUP mortality:
No treatment-related mortality occurred.

F1-PUP clinical signs:
No treatment-related clinical signs were noted.

Piloerection was noted in one pup at 2500 ppm and three pups at 10000 ppm 1-7 days after weaning. Because of the low incidence these findings were considered to be of no toxicological relevance. In addition, 8 pups of one female (at 10000 ppm) were found to be very small and dehydrated. However, as this finding concerned only one litter, this effect was also considered to be fortuitous.

Incidental clinical symptoms consisted of cold, small and/or pale appearance, no milk in the stomach, blue spots on the neck, chromodacryorrhoea, broken or wounded tail, and alopecia and scabs at several body parts.

F1-PUP body weights:
From Day 4 of lactation onwards, pups of the 10000 ppm group showed lower body weights when compared to the control group at increasing severity with time (from 92% of the body weights of control pups on Day 4 to 67% on Day 28). At birth, body weights were in the same range as those of the control animals.

F1-PUP Vaginal opening and balanopreputial separation:
The day of vaginal opening (VO) and balanopreputial separation (BPS) was delayed in pups of the high dose group (BPS: Day 48.5 at 10000 ppm compared to Day 42.9 in the control group; VO: Day 39.0 at 10000 ppm compared to Day 34.8 in the control group). Based on the fact that the mean body weights on the day of measurement for this group were lower than controls, these delays were considered secondary to the reduced body weights.

F1-PUP Macroscopic findings:
One pup (at 10000 ppm) showed blackbrown discolouration of the liver at necropsy. Although this finding was observed in only one pup, this finding correlated with the findings observed in the F0- and F1-generation and F2-pups and was therefore considered to be treatment-related.

Other macroscopic findings comprised small appearance, no milk in the stomach, pelvic dilation of the kidney and/or wound on the tail. For dead pups, in addition, cannibalism was noted. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

F1-PUP Organ weights:
At necropsy, decreased body weights were accompanied by decreased absolute spleen and thymus weights at 10000 ppm. In addition, decreased brain weights were noted at 10000 ppm, but together with an increased brain to body weight ratio this was considered to be related to the decreased body weights.


F2-PUP DEVELOPMENT
F2-PUP Mortality:
No treatment-related mortality occurred.

One pup (at 2500 ppm) was killed in extremis on Day 1 of lactation. This death was not considered to be related to treatment.

F2-PUP Clinical signs:
There was an increased incidence of small appearance of pups at 10000 ppm. No other treatment-related clinical signs were noted.

Other incidental clinical symptoms consisted of small appearance at low incidence in other groups, cold appearance, dry skin, no milk in stomach, purple to blue discolouration or staining of several body parts, scabs on the back, wound paw and red spots on the head. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

F2-PUP Body weights:
From Day 1 of lactation onwards, pups of the 10000 ppm group showed lower body weights when compared to the control group at increasing severity with time (from 93% of the body weights of control pups on Day 1 to 69% on Day 21).

From Day 14 of lactation onwards, pups of the 2500 ppm group also showed slightly lower body weights when compared to the control group (92% of the body weights of control pups on Days 14 and 21).

F2-PUP Anogenital distance:
No treatment-related effect on the anogenital distance was noted up to 10000 ppm.

F2-PUP Macroscopic findings:
At 10000 ppm, dark red to black brown discolouration of the liver was noted in 90 pups (14 litters) at necropsy on Day 21 of lactation. At 10000 ppm, also enlarged renal lymph nodes were noted in 7 pups (2 litters).

Other macroscopic findings of pups comprised small appearance, no milk in the stomach and hernia diaphragmatica of the lobus sinister medius of the liver. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

F2-PUP Organ weights:
At 10000 ppm, decreased body weights were accompanied by decreased absolute spleen and thymus weights. In addition, decreased brain weights were noted at 10000 ppm, but together with an increased brain to body weight ratio this was considered to be related to the decreased body weights.

At 2500 ppm, slightly decreased body weights were noted at necropsy. No other effects were noted.

F2-PUP Microscopic examination:
The following microscopic findings in the F2-pups were considered to be related to treatment with the test substance:
- Panlobular hepatocellular hypertrophy in the liver of 46/46 males and 46/46 females (minimal to slight) at 10000 ppm,
- Biliary/peribiliary brown pigment in 46/46 males and 46/46 females (minimal to moderate) at 10000 ppm and in 17/19 males and 17/19 females at 2500 ppm (minimal to slight),
- Bile duct proliferation in 46/46 males (minimal to moderate) and 46/46 females (minimal to slight) at 10000 ppm and in 9/19 males and 10/19 females (minimal) at 2500 ppm,
- Peribiliary inflammation in 14/46 males (minimal) and 7/46 females (minimal) at 10000 ppm,
- Hyperplasia of the renal lymph node lymphoid in 1/2 males (minimal) and 5/5 females (minimal) at 10000 ppm.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
625 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects on body weight; organ weights; histopathology.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
625 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects on body weight; organ weights; histopathology.
Reproductive effects observed:
not specified
Conclusions:
Treatment of male and female Wistar Han rats with Trigonox 301 at dietary dose levels of 625, 2500 and 10000 ppm revealed F0- and F1-parental toxicity at 2500 and 10000 ppm, based on reductions in body weight gain and food intake, changes in organ weights and histopathological changes, especially in the liver and kidneys. No reproduction toxicity was observed up to 10000 ppm for both generations.
In the F0- and F1-offspring, reductions in body weight gain were noted at 2500 and 10000 ppm, and a slight reduction in the mean number of pups, and changes in organ weights at 10000 ppm. Histopathological liver and kidney changes, similar to those observed in the parents, were also noted at 2500 and 10000 ppm in the F1-offspring (F2- generation). No teratogenic effects were observed up to 10000 ppm.
Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was 625 ppm; the NOAEL for developmental toxicity was also 625 ppm. However, toxicity in the pups was seen in the presence of clear parental toxicity. The reproduction NOAEL was established as being at least 10000 ppm.
When corrected for the mean value of recovery, the NOAEL of 625 ppm corresponded to 29-31 mg/kg bw/day for the F0 premating period and 32-36 mg/kg bw/day for the F1 premating period and the NOAEL of at least 10000 ppm corresponded to 488-512 mg/kg bw/day for the F0 premating period and 572-591 mg/kg bw/day for the F1 premating period (male-female values).
Executive summary:

Title

A two-generation reproduction toxicity study of Trigonox 301 in rats by dietary administration.

 

Guidelines

The study procedures described in this report were based on the following guidelines:

1) Organisation of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 416, Two-Generation Reproduction Toxicity Study, January 2001.

2) TheEnvironmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3800, Reproduction and Fertility Effects, August 1998.

3) European Community (EC), Commission directive 2004/73/EC, Part B, Methods for the determination of toxicology, B.35:"Two-generation reproduction toxicity study", EC Publication No. L152, April 2004.

 

Rationale for dose levels

The dose levels were based on a 14-day range finding study (NOTOX Project 488325) in which rats were exposed to 625, 2500 and 10000 ppm by dietary administration. For the current study the same dose levels were selected based on slight toxicity observed at 10000 ppm (see also Appendix 9).

Study outline

The purpose of this study was to assess the effect of Trigonox 301 on male and female reproductive performance, such as gonadal function, estrous cycle, mating behaviour, conception, parturition, lactation, weaning, and on the growth and development of the offspring when administered during two complete reproductive cycles. The study may also provide information of the test substance on developmental toxic effects, such as neonatal morbidity, mortality, behaviour and teratogenesis and to serve as a guide for subsequent tests.

 

After acclimatisation, four groups of twenty-four male and twenty-four female Wistar Han rats were exposed by dietary administration to the test substance at 0, 625, 2500 and 10000 ppm.

The F0-generation was exposed for a minimum of 70 days prior to mating and continuing until euthanasia. The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 70 days prior to mating and continuing until euthanasia. The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.

 

Evaluated parameters

The following parameters were evaluated: mortality, clinical signs, body weights, food consumption, reproduction processes, observations of the offspring, macroscopy, organ weights, and histopathology. Chemical analyses of diets were conducted four times during the study to assess accuracy and homogeneity and once to assess stability. In addition, random samples from all diet preparations of Weeks 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, 29, 31 and 35 were taken and analyzed for accuracy (Groups 1-4). The reason for this was that the accuracies of the diets were below the target concentrations of 80-120%. The analytical method used was considered to be the most optimal.

RESULTS

 

Dietary analyses

Accuracies were determined for all prepared diets. The reason for this was that the accuracies of the diets were below the target concentrations of 80-120%. The analytical method used was considered to be the most optimal. The mean recoveries of the procedural recovery samples were in the range of 64% - 108%. In consultation with the Sponsor (also based on re-analysis of the samples, see Appendix 13 and 14), it was decided that the results of the procedural recovery samples were applied to correct the analysed concentrations of the test samples in order to minimize the effect of the analytical method on the results of the test samples. The mean accuracies of the diets of Groups 2, 3 and 4 after correction were in the range of 45% - 76%. Low accuracy values were also observed in the 14-day pilot study (NOTOX project 488325).

 

The coefficients of variation on the homogeneity of the diets were in the range of 2.9% - 14%. The relatively high values of > 10% were accepted since no tendency from top to bottom was observed. Therefore, it was considered that the diets were homogenous. Formulation analysis further showed that the formulations were stable for at least 22 days at room temperature.

The following changes were considered to be related to treatment:

F0-GENERATION

 

at 625 ppm (Group 2):

PARENTAL, REPRODUCTIONDEVELOPMENTAL TOXICITY / TOXICITY IN F1-PUPS:

No findings.

at 2500 ppm (Group 3):

PARENTAL TOXICITY:

-      Macroscopic abnormalities in the liver (discolouration), kidney (discolouration), adrenal gland (discolouration) and in the renal lymph nodes (discolouration and enlargement) in a few animals.

-      Organ weight changes in the liver of males.

-      Microscopic findings consisted of brown pigment (liver, kidneys and lymph nodes), peribiliary inflammation (liver), bile duct proliferation, lymphoid hyperplasia (lymph nodes) and macrophage infiltration (lymph nodes) in both males and females.

 

REPRODUCTIONDEVELOPMENTAL TOXICITY / TOXICITY IN F1-PUPS:

No findings.

 

at 10000 ppm (Group 4):

PARENTAL TOXICITY:

-      Decreased body weights, body weight gain and food consumption during the treatment period.

-      Macroscopic abnormalities in the liver (discolouration and enlargement), kidney (discolouration), adrenal gland (reduced size and discolouration), renal and hepatic lymph nodes (discolouration and enlargement) and in the spleen (enlargement). One or more of these findings were noted in a high number of animals.

-      Organ weight changes in the liver, kidneys, adrenal glands, spleen, testes, epididymides, ovaries and uterus.

-      Microscopic findings consisted of brown pigment (liver, kidneys and lymph nodes), centrilobular hypertrophy (liver), peribiliary inflammation (liver), bile duct proliferation, lymphoid hyperplasia (spleen and lymph nodes) and macrophage infiltration (lymph nodes) in both males and females. The observed brown pigment in the liver contained neither iron nor bile pigments.

 

REPRODUCTION TOXICITY:

No findings.

 

DEVELOPMENTAL TOXICITY / TOXICITY IN F1-PUPS:

-      A slight decrease in the mean number of living pups at first litter check.

-      Lower body weights for male and female pups.

-      Macroscopic abnormalities in the liver (discolouration) in one pup.

-      Organ weight changes in spleen and thymus.

 

F1-GENERATION

 

at 625 ppm (Group 2):

PARENTAL TOXICITY:

Microscopic findings consisted of brown pigment (liver, kidney and lymph nodes), centrilobular hypertrophy, bile duct proliferation and peribiliary inflammation in a few animals. These changes were seen at a very low incidence and minimal severity. Moreover, a few of these changes (e.g. peribiliary inflammation and pigment in the kidney) were also observed in animals of the control group at comparable incidence and degree. The effects noted at 625 ppm were of minimal nature and did not indicate an adverse effect on functional integrity.

 

REPRODUCTIONDEVELOPMENTAL TOXICITY / TOXICITY IN F2-PUPS:

No findings.

 

at 2500 ppm (Group 3):

PARENTAL TOXICITY:

-      Decreased body weights during lactation.

-      Macroscopic abnormalities in the liver (discolouration), kidney (discolouration) and adrenal gland (discolouration) in a few animals and abnormalities renal and/or pancreatic lymph nodes (discolouration and enlargement) in a high number of animals.

-      Organ weight changes in the liver, spleen and uterus.

-      Microscopic findings consisted of brown pigment (liver, kidneys and lymph nodes), centrilobular hypertrophy (liver), peribiliary inflammation (liver), bile duct proliferation, lymphoid hyperplasia (lymph nodes) and macrophage infiltration (lymph nodes) in both males and females.

 

REPRODUCTION TOXICITY:

No findings.

 

DEVELOPMENTAL TOXICITY / TOXICITY IN F2-PUPS:

-      Slightly lower body weights forF2-pups.

-      Microscopic findingsF2-pupsconsisted of brown pigment (liver) and bile duct proliferation.

 

at 10000 ppm (Group 4):

PARENTAL TOXICITY:

-      Decreased body weights, body weight gain and food consumption during the treatment period.

-      Macroscopic abnormalities in the liver (discolouration and enlargement), kidney (discolouration), adrenal gland (reduced size and discolouration) and in renal, pancreatic and/or hepatic lymph nodes (discolouration and enlargement) in a high number of animals.

-      Organ weight changes in the liver, kidneys, spleen, testes, epididymides, seminal vesicles and uterus.

-      Microscopic findings consisted of brown pigment (liver, kidneys and lymph nodes), centrilobular hypertrophy (liver), peribiliary inflammation (liver), bile duct proliferation, lymphoid hyperplasia (spleen and lymph nodes) and macrophage infiltration (lymph nodes) in both males and females.

 

REPRODUCTION TOXICITY:

No findings.

 

DEVELOPMENTAL TOXICITY / TOXICITY IN F2-PUPS:

-      A slight decrease in the total and mean number of living pups at first litter check.

-      Increased incidence ofF2-pups with a small appearance.

-      Lower body weights forF2-pups.

-      Organ weight changes in the spleen and thymus.

-      Dark red to black brown discolouration of the liver in a high number ofF2-pupsand enlarged renal lymph nodes in a few pups.

-      Microscopic findings inF2-pupsconsisted of brown pigment in the liver, panlobular hepatocellular hypertrophy, peribiliary inflammation, bile duct proliferation andlymphoid hyperplasia in the renal lymph nodes.

 

CONCLUSION

Treatment of male and female Wistar Han rats with Trigonox 301 at dietary dose levels of 625, 2500 and 10000 ppm revealed F0- and F1-parental toxicity at 2500 and 10000 ppm, based on reductions in body weight gain and food intake, changes in organ weights and histopathological changes, especially in the liver and kidneys. No reproduction toxicity was observed up to 10000 ppm for both generations.

 

In the F0- and F1-offspring, reductions in body weight gain were noted at 2500 and 10000 ppm, and a slight reduction in the mean number of pups, and changes in organ weights at 10000 ppm. Histopathological liver and kidney changes, similar to those observed in the parents, were also noted at 2500 and 10000 ppm in the F1-offspring (F2- generation).

 
Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was 625 ppm; the NOAEL for developmental toxicity was also 625 ppm. However, toxicity in the pups was seen in the presence of clear parental toxicity. The reproduction NOAEL was established as being at least 10000 ppm.

 

When corrected for the mean value of recovery, the NOAEL of 625 ppm corresponds to 398 ppm (29-31mg/kg bw/day for the F0premating period and 32-36 mg/kg bw/day for the F1premating period) and the NOAEL of 10000 ppm corresponds to 6630 ppm (488-512 mg/kg bw/day for the F0premating period and 572-591 mg/kg bw/day for the F1premating period) (male-female values).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the 2 -generation study, the parental No Observed Adverse Effect Level (NOAEL) was 625 ppm; the NOAEL for developmental toxicity was also 625 ppm. However, toxicity in the pups was seen in the presence of clear parental toxicity. No teratogenic effects were observed. The reproduction NOAEL was established as being at least 10000 ppm. When corrected for the mean value of recovery, the NOAEL of 625 ppm corresponded to 29 -31mg/kg bw/day for the F0 premating period and 32 -36 mg/kg bw/day for the F1 premating period and the NOAEL of of at least 10000 ppm corresponded to 488 -512 mg/kg bw/day for the F0 premating period and 572 -591 mg/kg bw/day for the F1 premating period (male-female values).


Effects on developmental toxicity

Description of key information
A well conducted developmental toxicity was available in rabbits
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - March 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
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: 20.0-
21.8°C), a relative humidity of 30 - 70% (actual range: 39 - 64%) and 12 hours artificial light and
12 hours darkness per day.
Accommodation
Females were individually housed in labelled cages with perforated floors (Techniplast, Italy,
dimensions 75 x 70 x 45 cm, or Ebeco, Germany, dimensions 67 x 62 x 55 cm).
Diet
Free access to pelleted diet for rabbits (K-H from SSNIFF® Spezialdiaten GmbH, Soest,
Germany). Results of analyses for nutrients and contaminants of each batch were examined
and archived. Copies of the certificates are enclosed in Appendix 10 of this report.
In addition, hay (Tecnilab-BMI BV, Someren,the Netherlands) was provided three times a week.
Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and
archived.
Analysis of diet and water did not reveal any findings that were considered to have affected
study integrity.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Oral gavage, using a plastic catheter attached to a plastic
disposable syringe.
Formulations were placed on a magnetic stirrer during dosing.
Once daily for 7 days per week, approximately the same time each
day with a maximum of 4 hours difference between the earliest and
latest dose.
Female 50 was not dosed on Day 21 post-coitum as this animal
showed rapid breathing on this day. Females 76 and 77 were not
dosed on Day 26 post-coitum due to bad health. Female 48 was
not dosed on Day 22 post-coitum as this animal showed rapid
breathing.
1 mL/kg body weight. Actual dose volumes were calculated
according to the latest body weight.
From day 7 to day 28 post-coitum, inclusive.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC-MS
Details on mating procedure:
Artificial insemination (number of donor bucks not indicated)
Duration of treatment / exposure:
Day 7 up to and including Day 28 post coitum (day of insemination = Day 0)
Frequency of treatment:
Daily
Duration of test:
Animals were killed on Day 29
No. of animals per sex per dose:
12 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups of twenty-three (control group) or twenty-four (treatment groups) New Zealand White rabbits were inseminated (Day 0 post-coitum) and exposed by oral gavage to 0, 30, 100 and 300 mg/kg TRIGONOX 301 from Day 7 to 28 post-coitum. Groups 1, 2, 3 and 4 consisted of 17,19,22 and 18 pregnant animals, respectively. The numbers of litters available for morphological evaluation were 16, 19, 20 and 12 in the control, 30, 100 and 300 mg/kg/day, groups respectively. Females were checked daily for the presence of clinical signs. Body weight and food consumption of females was determined at periodic intervals and from February 15th onwards, water consumption was measured daily. Formulation analysis was performed on prepared dosing solutions. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horns were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live/dead fetuses and embryo-fetal deaths, the weight of each fetus, fetal sex and externally visible fetal macroscopic abnormalities. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin' fixative and subsequently sliced, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations. Analyzes demonstrated accuracy, homogeneity and stability of dose preparations.
Maternal examinations:
Mortality I Viability: At least twice daily. Animals showing pain, distress or discomfort,
which was considered not transient in nature or was likely to
become more severe, were sacrificed for humane reasons based
on OECD guidance document on humane endpoints
(ENV/JM/MONOI (2000)7). The time of death was recorded as
precisely as possible.

Clinical signs: At least once daily from day 0 post-coitum onwards. The time of
onset, degree and duration were recorded. All symptoms were
graded according to fixed scales:
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
A different project number (NOTOX Project 486690) was used for
recording of clinical signs.
Cage debris was examined to detect abortion or premature birth.

Body weights: Days 0, 4 and 7-29 (daily) post-coitum.

Food consumption: Days 0-4, 4-7,7-10, 10-13, 13-16, 16-20,20-23,23-26 and 26-29
post-coitum.

Water consumption: From study initiation, water consumption was visually assessed.
From 15 February 2008 onwards, water consumption was
determined for all animals due to a possible treatment related
effect on water consumption.

Necropsy: All animals were subjected to an examination post-mortem. All animals surviving to day 29 postcoitum,
all moribund animals and all animals showing abortion or premature delivery were
euthanised by intravenous injection of pentobarbital (approx. 1 ml/kg Euthesate®; Ceva Sante
Animale BV, Maassluis, The Netherlands) immediately prior to necropsy.

Organ weights: The weight of the liver, kidneys and terminal body weight were recorded from all surviving
females on the scheduled day of necropsy. A different project number (NOTOX Project 486690)
was used for recording of organ weights.

Histopathology: The following slides were examined by a pathologist:
The liver and kidneys of the first ten pregnant females of Groups 1 and 4.
The liver of Female 58 (Group 3), as this female showed accentuated lobular pattern of the
liver at macroscopic examination (this finding was also noted for five animals of the high
dose group).
Due to treatment related changes in the liver at the high dose group, the liver of the first ten
pregnant females of Groups 2 and 3 were examined.
All abnormalities.
All abnormalities were described and included in the report. An attempt was made to correlate
gross observations with microscoRic findings.

Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and
examined as quickly as possible to determine:
- The number of corpora lutea (ovaries in situ)
- The weight of the gravid uterus
- The number and distribution of live and dead foetuses
- The number and distribution of embryo-foetal deaths
- The number of former implantation sites
- The weight of each live foetus
- The sex of each foetus (during further foetal examination)
- Externally visible macroscopic foetal abnormalities.
Fetal examinations:
External, visceral and skeletal foetal findings were recorded as developmental variations or
malformations. See Appendix 3 for the details of all foetal examination methods.
External:
Each viable foetus was examined in detail and weighed. All live foetuses were euthanised by
subcutaneous injection of 0.1 ml pentobarbital (Euthesate®; Ceva Sante Animale BV,
Maassluis, The Netherlands) in the area between the scapulas. The crown-rump length of late
resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a
gross external examination performed (if possible) and the tissue was discarded.
Visceral (Internal):
Foetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The
thoracic and abdominal cavities were opened and dissected using a technique described by
Stuckhardt and Poppe (Stuckhardt, 1984). This examination included the heart and major
vessels. The sex of all foetuses was determined by internal examination.
The heads were removed from approximately one-half of the foetuses in each litter and placed
in Bouin's solution (Klinipath, Duiven, The Netherlands) for subsequent processing and softtissue
examination using the Wilson sectioning technique (Wilson, 1965). After examination, the
tissues were stored in neutral phosphate buffered 4% formaldehyde solution (Klinipath, Duiven,
The Netherlands). The heads from the remaining one-half of the foetuses in each litter were
examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in
identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for
subsequent examination of skeletons.
Skeletal:
Each eviscerated foetus, following fixation in alcohol, was macerated in potassium hydroxide
(Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The
Netherlands) by a method similar tothat described by Dawson (Dawson, 1926). The skeletal
examination was made following this procedure. The specimens were archived in glycerin
(Klinipath, Duiven, The Netherlands) with bronopol (Merck, Darmstadt, Germany) as
preservative.
Statistics:
The following statistical methods were used to analyze the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett,
1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be
assumed to follow a normal distribution.
The Fisher Exact-test (Fisher 1950) 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.

The following interpretation was performed for fetal pathology data:
All statistical tests were performed using appropriate computing device programs. Analyses
were conducted using two-tailed tests (except as noted otherwise) for minimum significance
levels of 1 % and 5%, comparing each compound-treated group to the control group. Each mean
was presented with the standard deviation (S.D.) and the number of animals (N) used to
calculate the mean. Due to the different rounding conventions inherent in the types of software
used, the means and standard deviations on the summary and individual tables may differ by ±
1 in the last significant figure. Where applicable, the litter was used as the experimental unit.
Mean litter proportions (percent per litter) of total fetal malformations and developmental
variations (external, visceral, skeletal and combined), and each particular external, visceral and
skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA
test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed
statistically significant (p<0.05) intergroup variance, Dunn's test (Dunn, 1964) was used to
compare the compound-treated groups to the control group.
Indices:
Pre-implantation loss (Number of corpora lutea - number of implantation sites) x 100
Number of corpora lutea
Post-implantation loss (Number of implantation sites - number of live foetuses) x 100
Number of implantation sites
The following calculations were performed for fetal developmental findings:
The fetal developmental findings were summarized by: 1) presenting the incidence of a given
finding both as the number of fetuses and the number of litters available for examination in the
group; and 2) considering the litter as the basic unit for comparison and calculating the number
of affected fetuses in a litter on a proportional basis
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At the high dose group (300 mg/kg body weight/day), severe toxicity was noted. Eight rabbits were killed in extremis before planned necropsy. Six of these animals showed an early delivery. All females showed clinical signs, severe body weight loss, and reduced food consumption and water consumption. One additional female of the high dose group showed an early delivery on the day of planned necropsy. Clinical signs observed at the high dose group consisted of lethargy, hunched posture, slow breathing, pale appearance, piloerection, brown discolouration of the urine, pale faeces, reduced faeces production, lean appearance and hypothermia. Animals treated at 300 mg/kg showed severe body weight loss, severely reduced food consumption, and reduced water consumption.
Treatment related macroscopic findings for the liver and gallbladder were observed at 300 mg/kg. Six animals were emaciated at necropsy. Liver findings consisted of accentuated lobular pattern, enlargement, hardening and black-brown discoiouration. Gallbladder findings consisted of black discoloured contents and a gelatinous gallbladder. Increased liver weights (absolute and relative) and kidneys weights (relative) were noted at the high dose group. Histopathological examination revealed severely affected livers with hypertrophic hepatocytic changes and proliferative bile channel changes.
At the intermediate dose group (100 mg/kg body weight/day), similar but less severe effects on the liver were noted. These consisted of accentuated lobular pattern for one animal and minimal centriacinar hepatocytic hypertrophy for three animals.
No maternal toxicity was observed at the low dose group (30 mg/kg);
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No effects on pre- and post implantation loss, litter size and sex ratio were noted. Significantly reduced body weights of fetuses were noted at the high dose group (300 mg/kg). This was considered to be related to the decreased maternal body weight at this dose level. An increase in the mean litter proportion of fetuses with unossified metacarpais (statistically significant) and unossified sternebrae nos. 5 and/or 6 were noted at a dose level of 300 mg/kg/day. This delayed ossification, however, was considered secondary to the fetal body weight reduction noted at this dose level.
There were no test substance related effects on fetal morphology in the 30 and 100 mg/kg/day group.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for TRIGONOX 301 was established as being 30 mg/kg body weight/day. The NOAEL for embryo/fetal developmental toxicity, which was considered to be secondary to the documented maternal toxicity, was 100 mg/kg body weight/day.
Executive summary:

Four groups of twenty-three (control group) or twenty-four (treatment groups) New Zealand White rabbits were inseminated (Day 0 post-coitum) and exposed by oral gavage to 0, 30, 100 and 300 mg/kg TRIGONOX 301 from Day 7 to 28 post-coitum. Groups 1, 2, 3 and 4 consisted of 17,19,22 and 18 pregnant animals, respectively. The numbers of litters available for morphological evaluation were 16, 19, 20 and 12 in the control, 30, 100 and 300 mg/kg/day, groups respectively. Females were checked daily for the presence of clinical signs. Body weight and food consumption of females was determined at periodic intervals and from February 15th onwards, water consumption was measured daily. Formulation analysis was performed on prepared dosing solutions. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horns were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live/dead fetuses and embryo-fetal deaths, the weight of each fetus, fetal sex and externally visible fetal macroscopic abnormalities. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin' fixative and subsequently sliced, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations. Analyzes demonstrated accuracy, homogeneity and stability of dose preparations.

Maternal findings At the high dose group (300 mg/kg body weight/day), severe toxicity was noted. Eight rabbits were killed in extremis before planned necropsy. Six of these animals showed an early delivery. All females showed clinical signs, severe body weight loss, and reduced food consumption and water consumption. One additional female of the high dose group showed an early delivery on the day of planned necropsy. Clinical signs observed at the high dose group consisted of lethargy, hunched posture, slow breathing, pale appearance, piloerection, brown discolouration of the urine, pale faeces, reduced faeces production, lean appearance and hypothermia. Animals treated at 300 mg/kg showed severe body weight loss, severely reduced food consumption, and reduced water consumption. Treatment related macroscopic findings for the liver and gallbladder were observed at 300 mg/kg. Six animals were emaciated at necropsy. Liver findings consisted of accentuated lobular pattern, enlargement, hardening and black-brown discolouration. Gallbladder findings consisted of black discoloured contents and a gelatinous gallbladder. Increased liver weights (absolute and relative) and kidneys weights (relative) were noted at the high dose group. Histopathological examination revealed severely affected livers with hypertrophic hepatocytic changes and proliferative bile channel changes. At the intermediate dose group (100 mg/kg body weight/day), similar but less severe effects on the liver were noted. These consisted of accentuated lobular pattern for one animal and minimal centriacinar hepatocytic hypertrophy for three animals . ................... No maternal toxicity was observed at the low dose group (30 mg/kg). Developmental findings No effects on pre- and post implantation loss, litter size and sex ratio were noted. Significantly reduced body weights of fetuses were noted at the high dose group (300 mg/kg). This was considered to be related to the decreased maternal body weight at this dose level.

An increase in the mean litter proportion of fetuses with unossified metacarpals (statistically significant) and unossified sternebrae nos. 5 and/or 6 was noted at a dose level of 300 mg/kg/day. This delayed ossification, however, were considered secondary to the fetal body weight reduction noted at this dose level. There were no test substance related effects on fetal morphology in the 30 and 100 mg/kg/day group.

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
100 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Based on the results in the prenatal developmental toxicity study in rabbits, the NOAEL for embryo/fetal developmental toxicity (these effects were considered to be secondary to the documented maternal toxicity) was 100 mg/kg body weight/day.

No effects on pre- and post implantation loss, litter size and sex ratio were noted. Significantly reduced body weights of fetuses were noted at the high dose group (300 mg/kg). This was considered to be related to the decreased maternal body weight at this dose level.

An increase in the mean litter proportion of fetuses with unossified metacarpals (statistically significant) and unossified sternebrae nos. 5 and/or 6 was noted at a dose level of 300 mg/kg/day. This delayed ossification, however, were considered secondary to the fetal body weight reduction noted at this dose level. There were no test substance related effects on fetal morphology in the 30 and 100 mg/kg/day group.

Justification for classification or non-classification

Based on the results of the developmental toxicity study in rabbits and the 2 -generation study in rats, no classification is needed for fertility and developmental toxicity.