Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From Feb. 12, 1985 to Nov. 18, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Him:OFA (Sprague-Dawley) SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: The rats were obtained from research Institute for Experimental Animal Breeding, A-2325 Himberg- Age at study initiation: Initial parental animals were 8 wk old at study initiation. F1 generation was placed on treatment at weaning (at approx. 21 d of age).- Weight at study initiation: The weight range of the animals at study initiation were:- Parental (P) Generation: Males: The group means ranged from 209.15 ± 21.03 to 222.53 ± 30.22 g; Females: The group means ranged from 176.57±12.83 to 182.58±10.88 g; - F1 generation: Males: the group means ranged from 59.03±13.83 to 67.89+/-8.24 g; Females: the group means ranged from 55.40±11.63 to 63.86+/-8.41 g- Fasting period before study: Not reported- Housing: The animals were housed in Makrolon type III cages with wire grid covers. The cage allocation were as follows:Before mating: 2 male or 2 female per cageAfter mating: 2 male or 1 female per cageFrom weaning and reduction of pups: 2 male or 2 female per cage The housing of the animals on the cage racks was ordered, two groups per rack (one group per side of frame). The position of the racks in the room was swapped each day in a cyclic manner.- Diet: Altromin 1314 ff, 10 kGy gamma-irradiated (for parental generation), Altromin softwood granulate, autoclaved (for litters) (ad libitum) - Water: Tap water from Makrolon watering bottles, (ad libitum)- Acclimation period: 1 wkENVIRONMENTAL CONDITIONS- Temperature: 22°C (on an average)- Humidity: 60% (mostly), with short-term peaks between 30% and 70%.- Air changes: 15 per hour- Photoperiod:12 h light/12 h dark cycleIN-LIFE DATES: From: Feb. 27, 1985 To: Nov. 18, 1985
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(deionised, containing 50 µl of 25% ammonia per g test substance)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance solution in deionised water (containing 50 µl of 25% ammonia per g test substance) - Rate of preparation of dose (frequency): The test solution was freshly prepared daily immediately prior to the administration.- pH of dosing solutions: Approx. 6- Dose volume: 10 mL/kg, calculated in each case on the basis of the body weight last measured (for inseminated females, the body weight on the 6th d of gestation was used for the calculation from the 6th d of gestation until the day of birth).VEHICLE (Deionized water)- Justification for use and choice of vehicle: Not reported- Concentration in vehicle: 0.5, 1.5 and 4.5 mg/mL for the dose levels of 5, 15 and 45 mg/kg bw- Amount of vehicle: 10 mL solution per kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1- Length of cohabitation: Until proof that insemination had take place, but maximally for 3 wks- Proof of pregnancy: Sperm in the vaginal smear or when a vaginal plug was found, the day was referred to as Day 0 of pregnancy. Vaginal smear with a cotton wrapped, moistened stick and smearing on a microscope slide was used for the evaluation of the estrous cycle phase and determination of sperm.- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Not reported- Further matings after two unsuccessful attempts: Not reported- After successful mating each pregnant female was caged: Individually in Makrolon type III cages with wire grid covers- Other: Pairing of siblings in the F1 generation was avoided by the assignment mode at weaning. There was one pairing of siblings in the low dose group due to a mistake.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Parental Generation:- Males: From the first experimental day until mating for 70 d, during the entire mating period and further until exclusion from the experiment.- Females: Immediately prior to mating for 14 d, during the entire mating period, gravidity and lactation and further until exclusion from the experiment.F1 Generation:- Males and females from weaning (21st d after birth) for approximately 80 d until start of mating and further until exclusion from the experiment.
Frequency of treatment:
Once daily (7 x per week) in the a.m. The sequence of administration to the groups was altered daily in the cyclic manner.
Details on study schedule:
- F1 parental animals not mated until approx. 101 d after birth.- Selection of parents from F1 generation when pups were weaned, 21 d after birth: - Age at mating of the mated animals in the study: P generation: Approx. 18 wks; F1 generation Approx 14 wks
Remarks:
Doses / Concentrations:0, 5, 15 and 45 mg/kg bw Basis:actual ingested
No. of animals per sex per dose:
24 rats per sex per dose (Parental and F1 Generations)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dosage was chosen according to the results of several toxicological investigations of test substance in rats. This included determination of LD50, teratology study and subacute oral toxicity study. Details are provided in the study report.- Rationale for animal assignment: Random, using a method of random numbers with increasing body weights, males and females assigned separately, and also taking care to avoid the pairing of siblings in the subsequent mating.- Other: Count of experimental and lactation days.Experimental Day: The first experimental Day was the Day on which the test substance was administered to male animal for the first time.Lactation Days (Day after birth of pups): The Day on which pups were born was considered Day 1.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: Once daily- Cage side observations included: Special attention was given to the behavior during mating, to the care behavior of damDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Veterinary examinations of the animals were conducted on a case-by-case basis.BODY WEIGHT: Yes- Time schedule for examinations: Body weight was determined on receipt and once per week (Wednesday) from the first experimental day with the following exceptions: Inseminated females were evaluated on gestation Day 0, 6, 13 and 20 Dams on the Day 1, 4, 7 and 21 after birth of their pups. FOOD CONSUMPTION - Food consumption: yes; Food consumption was determined once weekly from Tuesday to Tuesday with the following exceptions: for inseminated female animals on gestation day 0-6, 6-13 and 13-20; for dams with live pups on the 1st-7th, 7th-14th and 14th-21st d after birth of the pups. No determination of feed consumption was carried out for male animals after insemination took place.- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
Estrous cyclicity (parental animals):
The cycle determination was carried out by vaginal cytology daily in the morning in all female animals from approx. 2 wks prior to the start of mating (P generation: from the 57th experimental day; F1 generation: from the 190th experimental day) until insemination took place. Vaginal smear with a cotton wrapped, moistened stick and smearing on a microscope slide was used for the evaluation of the estrous cycle phase and determination of sperm.
Sperm parameters (parental animals):
Weight of epididymis and testes were determined in fixed state. No other details on sperm parameters are provided in the study report.
Litter observations:
STANDARDISATION OF LITTERS- Performed on day 4 postpartum: Yes- Litter size was reduced to maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.PARAMETERS EXAMINED: The examination of pups as well as the sex determination and weighing were only carried out after the cleaning by the dam, i.e. possible only on the Day 2 after birth of the pups.The following parameters were examined in offspring: i) Examination before, during and after birth:- Potential external visible malformation and injuries.- Occurrence of milk intake by pups and care behavior by the dam- Number of live & dead pupsii) Number, sex and body weight of the pups after birth, and on Day 4, 7 and 21 after birthiii) Developmental stages of pups and behavioral test (From birth to weaning):- Ear folding: From Day 2 after birth, ears of pups were examined for unfolding. The number of pups with unfolded ears was recorded daily.- Eye opening: Separation of eyelids was observed and recorded approx. from Day 10 after birth.iv) Examinations on the day of weaning (21 d after birth):- Auricular reflex- Orientation reaction- Lifting test and grasp reflexDetails are provided in the study report.WEANING OF PUPS- All pups were weaned on Day 21 after birth.- 1 male and 1 female of the pups from each litter of parental generation was selected randomly.If a dam did not have atleast 1 male and 1 female pup, the shortage was adjusted by retaining additional pups from another litter of the same group.GROSS EXAMINATION OF DEAD PUPS: Yes: Until the Day 4, dead pups were litter-wise fixed in Bouin solution immediately after being discovered. Necropsy of these animals and those accumulating from reduction of pups on Day 4 (killed by carbon dioxide gas) was carried out under the stereo microscope. The abdominal and thoracic cavities were opened and the viscera examined. The cranium was then removed and sectioned in a manner similar to the technique of Wilson (longitudinal section along the jaws, at least 3 transverse sections through nasal cavity, eye and brain).
Postmortem examinations (parental animals):
SACRIFICE- Male animals: All surviving animals were killed and dissected on the 111th and 112th experimental d, respectively.- Maternal animals: All the animals were killed by CO2 after weaning and then dissected with special consideration of the reproductive organs. The female animals that were obviously not pregnant were killed and dissected on the 106th experimental day.GROSS NECROPSYSSpontaneously dying animals were dissected as soon as possible. If the waiting period was more than 2 h, interim storage was effected in the refrigerator. All sexual organs and other organs possibly noted to be altered were fixed. Weight of pituitary gland, epididymis, testes and ovaries was determined in fixed state.HISTOPATHOLOGY/ORGAN WEIGHTSApart from the organs that were noted at necropsy to be altered, following organs were examined histopathologically in parental animals of both generation with no live offspring, and in all parental animals of the control and high dose group: pituitary gland, mammary gland, vulva, vagina, cervix, uterus, tubes, ovaries, and respectively, penis, testis, epididymis, ducti deferentes, coagulation gland, prostate gland, and vesicular gland.
Postmortem examinations (offspring):
SACRIFICE- The F1 offspring not selected as parental animals were sacrificed at Day 4 of age.- All F2 offspring were sacrificed after weaning- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Necropsy of these animals was carried out under the stereo microscope. The abdominal and thoracic cavities were opened and the viscera examined. The cranium was then removed and sectioned in a manner similar to the technique of Wilson (longitudinal section along the jaws, at least 3 transverse sections through nasal cavity, eye and brain).- Older, spontaneously dying animals and weaned animals were dissected in the same way as the parental animals.- No histological examination of the organs of the pups or sampling for this purpose was carried out generally.
Statistics:
- Arithmetic means and standard deviations of all parameters (wherever meaningful) was calculated. The comparison of the means was preferentially conducted with the simple analysis of variance and subsequent Scheffe test. - If the pre-conditions of the simple analysis of variance were obviously not met, the H-test according to Kruskal and Wallis was used. - Counted events (e.g. the number of pregnant animals) were compared with the Chi squared test, in the case of low expected frequencies with Fisher's exact test.- The significance level was chosen in each case as p=0.05.
Reproductive indices:
See parental sections above
Offspring viability indices:
See offspring sections above.
Clinical signs:
no effects observed
Description (incidence and severity):
- One female in the high dose group died a t night between the 20th and the 21st lactation day (115th/116th experimental day). This animal died from the consequences of injuries due to the stomach tube during administration. - Crusted eyes, crusted nose, and local hair loss were observed again and again during the entire experiment in all groups. They were not associated with the effect of the test substance. - Yellow coloring of the ventral coat was de scribed in a few animals from the control and the low and mid dose groups on the 61st and 62nd day.Such discoloring is known historically and is due to urinary contamination. - Reddened and thickened auricles were found in some males from the 43rd (control), 70th (mid dose) and the 71st d (high dose), respectively, until the death of the animals. Redness and thickening of the auricles is known historically and was most likely caused independently of the substance by an incompatibility with and local allergic reactions to metal ear marks as used in this experiment. Minor, harmless bites were described in some animals from all groups. - Individual animals in both generations were described as nervous on some days. Particularly females were described as nervous between the Day 59 and 65 and between Day 73 and75
Body weight and weight changes:
no effects observed
Description (incidence and severity):
P generation: No statistical difference between dosed groups and the control group was observed at any time point. During gestation and the lactation period, the lowest body weights were observed in the control group. The differences to the other groups, however, were only marginal. The male animals clearly gained less weight than before between the 64th and the 106th experimental day in all groups. There was even a decrease in the body weight in the mid and high dose groups between the 64th and 71st and between the 99th and 106th experimental day, respectively. Apart from gravidity and the lactation period, such effects were not observed in females. The body weight gain measured from the start of administration, i.e. from the 1st experimental day for males and from the 57th day for females, until the start of the mating period (71st experimental day) was somewhat lower in females of the high dose group than in the control group. However, significant differences did not occur. Females lost weight between the 14th and 21st lactation day. Differences between the control and the dosed groups did not occur. F 1 generation: The weekly body weight gains in the middle ad highest dose group werehigher than the control group in males between Day 169 and Day 176 and in females between Day 183 and Day 190. From Day 204 to 209 only a minimal increase in body weight was observed in males and even a general decrease in females. A comparison of body weight gain between Day 134 and Day 209 (start of the mating period) does not show difference between the groups
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
FEED CONSUMPTIONP generation: Differences in feed consumption occurred in females prior to the application of the test substance. No further information was given on food consumption.F1 generation: There were marginal but significant differences between the control group and one test substance group between the 175th and the 182nd experimental day (males: mid dose higher than controls; females: low dose lower than controls). Since differences in feed consumption already occurred in females of the P generation prior to the application of the test substance, these two isolated findings were assessed as not related to the test substance
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
ORGAN WEIGHTS: - Males: In both P and F1 generation: no significant differences between the individual groups occurred, neither in the means of the absolute nor the relative organ weights of testis, epididymis and pituitary gland.- Female: The mean of the relative weight of the ovaries was significantly lower in the low dose group than in the control group (in the mid dose group, the relative weight of the ovaries was about equal to that in the low dose group but the difference to the control group was not significant due to the higher standard deviation in the mid dose group. This was considered a chance finding since the ovaries in the high dose group were even somewhat heavier than in the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
GROSS PATHOLOGY: Since the histopathological examination of the organs noted at necropsy to be altered largely only confirmed the necropsy findings, gross pathological findings were not mentioned in the report
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
HISTOPATHOLOGY:- Spontaneously deceased and prematurely killed animals: One high dose female in P generation died from respiratory and cardiovascular failure, manifested by serous hydrothorax and pulmonary oedema. The cause, dating back a few days, was probably an erroneous application of the test substance to the lung. This interpretation was supported by alveolar pigment deposits and peribronchial infiltrates. An influence of the test substance was not discernible in this death. Pulmonary oedema, blood aspiration of the lung and alveolar pigment deposits were found in 1 male animal in F1 generation pointing to an in jury by the stomach tube.- Parental animals without live pups despite proven insemination: No changes in any of the male partners of infertile pairs were detected, which in type and severity could have been the cause of a possible sterility. In the female partner, there was an altered endometrium in one control female, which was suggestive of hormonal disturbances. A small mammary fibroadenoma was found in one low dose female, which was of the type sporadically found in lactating rats. This tumor may thus also point to hormonal imbalances. The oedemas in one ovary of a high dose female and the dilated end ometrial glands were probably caused by a disturbed hormone balance as well. No changes were found in the remaining female partners of infertile pairs, which type and severity could have been the cause ofsterility.The type and distribution of these changes did not allow a conclusion of a test substance-related effect.- Parental animals of the control and the high dose group in both generations: A number of changes were detected in the histopathological examination of pituitary glands and reproductive organs from all Parental animals. However, in no case significant differences in the frequency and degree of severity was identified. The major findings obtained were as follows: In male animals. The alteration most often detected in the testes was a degeneration of individual tubules, which exclusively occurred in the tubules directly at the tunica albuginea. This often occurs spontaneously in the rat strain used and obviouslydoes not have an influence on the fertility. Only a single altered tubule per testis affected was mostly found.- A regional or also extensive atrophy of the germinal epithelium in the testis was observed in some cases. Even with these frequent, spontaneously occurring lesions, normal reproductive success occurred due to the remaining functional tubules.Interstitial oedemas and fibroses of the testes frequently occur spontaneously in the rat strain used. - The dilation of the lumina of the spermatic cords described in several cases should be related to an agonal ejaculation. Inflammatory alterations of the accessory sex glands, in particular the prostate, are often found in male rats without the fertility being affected- An inflammatory alteration of the preputial glands, mostly leading to abscesses, was repeatedly found.- In female animals : Most of the findings obtained for ovaries were related to differences in blood supply and differences inthe strength of lymphatic drainage, both of which in turn are related to the specific cycle phase. Therefore , they do not represent pathological changes.- Frequently, ovarian cysts occur spontaneously in the rat strain used. In this experiment, there were significantly more frequent in animals of the control group . As expected, almost all affected animals had a good reproductive success, the accumulation of abnormal findings in the control groups appeared to be due to chance.- A mammary tumor, a sporadic spontaneous occurrence in lactating rats, was found in one mid dose female.- The most frequent alteration of the uterus was a dilation of the lumen. This can be explained by abnormally pronounced characteristicsof the specific cycle phase. - An inflammation in the area of the vagina/vulva was described in individual cases. All other alterations described in female animals were isolated findings and spontaneous changes of no relevant significance.- The pituitary gland was examined histopathologically in both sexes, and only known, spontaneously occurring lesions were observed. The most frequency of these was cysts in different sites and, predominantly in the male animals, dilated lymphatic vessels. Organs examined additionally: In some cases, an auricular chondritis was found in animals, which must be attributed to an incompatibility with the ear marks.- A localized dermatitis occurred in one animal-Apart from focal proliferation of alveolar macrophages, which stood out as white subpleural foci, some chance finding were collected from the lungs examined in the context of these foci.- An adiponecrosis in the accessory tissue of the spermatic cord was found in one case.Overall, no influence of the test substance could be deduced from all findings gathered from these animals. In all cases, the type and frequency of occurrence conform to expectations, deduced also from historical data.
Other effects:
not specified
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
REPRODUCTIVE FUNCTION: ESTROUS CYCLE: P generation: Approx. 20% of the animals in the control and low and mid dose groups showed a normal sexual cycle. Only an irregular cycle or no cycle at all could be observed in the other animals and in the highest dose group. The difference between the groups was not statistically significant.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
REPRODUCTIVE FUNCTION: SPERM MEASURES: An increased release of immature cells was found in the testes in a couple cases which were identified as not treatment related. No significant difference between the individual groups occurred, neither in relative nor in absolute organ weights of testis and epididymus
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE- P generation: The time between the start of mating and insemination was a mean of 4.8-5.9 d. It decreased somewhat with increasing dose, but this was not statistically significant. The duration of gravidity averaged 22 d and was not influenced by the test substance. The fertility index, i.e. the number of pregnant animals as a percentage of the inseminated animals and therefore a measure of the fertility, was between 79 and 91 % and was also not influenced by the test substance.- F1 generation: Only one gravid dam (low dose) did not give birth to a single live pup. One dam (control) could not give birth due to a uterine rotation and was killed on gestation Day 26. The gestation index, i.e. the number of animals with live pups as a percentage of the number of pregnant animals, was thus below 100 % only in the low dose group, namely at 95% (Assuming that the one dam killed in the control group would have given birth to dead pups only, the gestation index in the control group would be below 100 % as well, namely 96%.). There was no significantdifference between the value of the low dose group and that of the control group. The fertility index in F1 generation was between 83% and 100% and was not influenced by the test substance.findings at birth were not made neither in P nor in F1 generation. Differences caused by the test substance in the behavior of the dams towards their pups or in lactation were not observed.
Key result
Dose descriptor:
NOAEL
Remarks:
(for Reproductive toxicity)
Effect level:
>= 45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
NOAEL
Remarks:
(maternal toxicity)
Effect level:
>= 45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Clinical signs:
no effects observed
Description (incidence and severity):
CLINICAL SIGNS: No group specific differences were observed in the pups. While the number of findings increased with dose, no statistically significant differences from the control group were detected. Non – significant changes in animal or of behavior such as subcutaneous haematomas, anemia, cyanosis and crushing and dispersal of feed were observed in animals from all groups, both in P and F1 generation
Mortality:
no mortality observed
Description (incidence):
VIABILITY: A breakdown by the number of live and dead pups per litter did not show substance specific differences either. The total number of pups per litter was not influenced by the test substance, neither for P nor for F1 generation.P generation: Some pups died between lactation days 1 and 4 in all groups: 2-6 per group. Only a few animals died after the 4th day, maximally 2 per group. No significant differences could be detected between groups. No significant difference could be detected between groups in the P generation.F1 generation: The number of pups deceased between the 1st and the 21st Day after birth in the middle dose group was significantly higher than in the control groups and the other dose groups.This difference was noted as not due to a generally higher fraction of deceased pups per litter in this group, rather to the extreme values from two litters.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
BODY WEIGHT: The body weight development in both generations was without noticeable difference. There were sporadically pups with a clearly lower body weight (<70% of the mean body weight in the litter) at birth. One and four such incidences were observed in F1 and F2 generation pups respectively. One animal (F2 generation) from one mid dose group had a clearly reduced body weight on the Day 14 and died on the Day 15. Ileus was found as the cause of death at necropsy. A relationship with the dosage did not exist.
Other effects:
no effects observed
Description (incidence and severity):
DEVELOPMENTAL STAGES AND BEHAVIORAL TEST OF THE PUPS:Ear unfolding occurred in all groups approx. between the Day 3 and 4 after birth and eye opening approx. on Day 16 after birth. Difference caused by the test substance didnot occur in the reaction and behavioral tests.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
>= 45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: maternal toxicity
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Description (incidence and severity):
SEXUAL MATURATION (OFFSPRING): F1 (as adults): Approx. 25-40% of the females in all groups had a normal sexual cycle, the others an irregular one or none at all. No significance was associated with this effect
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
ORGAN WEIGHTS (OFFSPRING):F1 (adults): In the males, no significant differences between the individual groups occurred, neither in the means of the absolute nor the relative organ weights of testis, epididymis and pituitary gland. In the females, the pituitary gland weights of the mid dose group were significantly higher than in the control group. The weight was about as low in the low dose group as in the control group. In the histopathological examination of the pituitary gland, no differences could be found between the control and high dose group, such as increased formation of cysts, adenomas etc. Therefore, this weight difference appeared rather incidental and at any rate had no effect on the fertility. The ovaries were generally heavier in F1 generation than in P generation. However, no difference between the groups was observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
GROSS PATHOLOGY:Spontaneously deceased pups: Individual cases of congenital lesions were found, such as uterine hypoplasia, serous pleura content, thickened pericardium, atria larger than expected, partial atrophy ofthe myocardium and unilateral anophthalmia, hydroureters and kidneys larger than expected. All of the se lesions were, in type and frequency, known as spontaneously occurring malformations and variations in the rat strain used.There were a number of different causes of death in the pups deceased from the Day 2 after birth: Heart defects, enteritis, pleurisy, anaemia, inguinal hernia and bleeding to death from the umbilical vessels were found, but no relation to a specific group or test substance exposure was discernible. The findings obtained from the spontaneously deceased pups did not permit any inference of an influence of the test substance.Pups excluded on Day 4 after birth: Mutilations of the tail tip were found relatively frequently but without relation to a specific group. The cause for this could not be identified. Furthermore, there were only irrelevant, minor traumas, which may have resulted from the maternal care behavior.Two malformed pups, one with unilateral testicular hypoplasia and one with brachygnathia inferior were found in one litter in the mid dose group in the F1 pups. Both malformations are known as rare spontaneous malformations in this rat strain. One stunted pup each was found in the control and mid dose F2 pups, but a cause for the developmental lag could not be found in these cases. In all these pups there was no discernible influence of the test substance.Pups excluded on Day 21 after birth: Only fouralterations were found, one case each of testicular hypoplasia, cryptorchism, tail trauma and high grade hydronephrosis. These changes are also known spontaneous changes without relation to test substance exposure.
Histopathological findings:
not examined
Description (incidence and severity):
HISTOPATHOLOGY: Older, spontaneously dying and weaned animals were dissected in the same way as parental animals. No histological examination of the organs of the pups or sampling for this purpose was carried out generally.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: development
Reproductive effects observed:
not specified
Conclusions:
Administration of p-Toluylenediamine sulphate by oral route in two generation study at 0, 5, 15 and 45 mg/kg bw/day was determined to have no adverse influence on female or male fertility and on rearing of pups in two generation at any of the dose levels tested.The NOAEL for reproductive effects, as well as maternal toxicity was determined to be >45 mg/kg bw/day.
Executive summary:

The two generation reproduction toxicity study of p-toluylenediamine sulphatewas determined followingOECD Guideline 416 (Two-Generation Reproduction Toxicity Study).

The study was designed to evaluate thetwo-generation reproduction toxicityof the p-Toluylenediamine sulphate inHim:OFA (Sprague-Dawley) SPFrats when administered orally at following concentrations:

0 (vehicle only), 5 (low dose group), 15 (Intermediate dose group) and 45 (High dose group) mg/kg bw/day.

The experimental animals (parental generation) were sourced from Research Institute for Experimental Animal Breeding, A-2325 Himberg at age of 8 wk and housed in Makrolon type III cages with wire grid covers at a ratio of 2 males or 2 females per cage before mating, one male and one female during mating, and one female or 2 males per cage after mating. From weaning and reduction of the pups, 2 males or 2 females were housed together per cage.  The housing was maintained at approx. 22°Cand 60% humidity with 15 air changes per hour. The animals were exposed to a 12 h light/12 h dark daily schedule and were allowed free access to food and water throughout the study.

The test substance was administered daily (7 x per week) as a solutionin deionised water (containing 50 µl of 25% ammonia per g test substance) by stomach tubeat a dosing volume of 10 mL/day. A similarly sized group of rats receiveddeionisedwater over the same time period and served as the control group.

In the parental generation (24 males and 24 females per dose group), the test substance was administered to males for 70 d and to females for 14 d prior to mating. The animals were subsequently monogamously mated with in the individual groups. Dams were further dosed until weaning of the pups and were then excluded from the experiment. In F1 generation 24 males and 24 females per dose group (randomly selected 1 male and 1 female of each litter) were retained and treated with test substance for 80 d starting on the Day 21 (at weaning) after birth. Subsequently, the animals were again monogamously mated within the groups. The offspring, (F2 generation) were kept for study until weaning age (21 d). 

The animals were observed for mortality and clinical signs once daily with special attention during mating, birth and development of pups. Body weight was determine on receipt and once per week with the following exceptions: Dams were weighed on Day 1, 4, 7, 14 and 21 after birth of their pups, and pups were weighed on the Day 1 or 2, 4, 7, 14 and 21 day after birth. Food consumption determined once weekly from Tuesday to Tuesday with the following exceptions: for inseminated female animals on gestation Day 0-6, 6-13 and 13-20; for dams with live pups on the Day 1- 7, 7-14 and 14-21 after birth of the pups. Vaginal smears for sexual cycle determination was carried out by vaginal cytology daily in the mornings in all female animals from approx. 2 wks prior to the start of mating (P generation: from the 57th experimental day; F1 generation: from the 190th experimental day) until insemination took place. Female animals of the P generation that were obviously not pregnant were killed and dissected on the 106th experimental day, those of the F1 generation on the 246th and 251st experimental day.

The examination of pups as well as the sex determination and weighing were carried out after the cleaning. Visible malformations and injuries were recorded, as were the occurrence of milk intake of the pups and the care behavior of the dam. The number of live and dead pups was recorded. The number, sex and body weight of the pups was determined on Day 4, 7, 14 and 21 after birth, at the same time as body weight determination. Ear folding, eye opening, auricular reflex, orientation reaction, lifting test and grasp reflex were examined in pups for positive response.

Spontaneously dying parental animals were necropsied as soon as possible. Excluded parental animals were killed by carbon dioxide gas and then dissected with special consideration of the reproductive organs. All sexual organs and all organs possibly noted at necropsy to be altered were fixed. Weights of the pituitary gland and the epididymis, as well as the testes and the ovaries, respectively, were determined in the fixed state. Apart from the organs that were noted at necropsy to be altered and then fixed, pituitary gland (all animals), females - mammary gland, vulva, vagina, cervix, uteus, tubes, ovaries; males - penis, testes, epididymes, ducti deferentes, coagulation gland, prostate gland, vesicular gland were examined histopathologically in all parental animals of both generations with no live offspring, as well as in all prenatal animals of the vehicle and the highest control group.

Body weight development, feed consumption and observation of parental animals did not show dose dependent differences between the groups, neither in the P nor in the F1 generation. Occasionally occurring anomalies could most probably be explained by external influences such as increased manipulation, noise etc and were independent of administration of dose.

The test substance did not show an influence, either on the P or on the F1 generation in relation to: duration and regularity of the female sexual cycle (; the fraction of inseminated animals; the time to; the fraction of gravid animals; the duration of gestation, gestation index, number of live/dead pups per litter when compared to the control group. Some anomalies were reported in mating, birth and rearing of the pups but were associated observations occurred probably as incidental isolated events.

Observations and measurements in pups of P and F1 generations until weaning did not show differences in body weight development in pups, sex ratio, development or behavior before, during and after birth, presence of different reflexes, reactions and physical developmental characteristics of pups and number of pups surviving until the 4th and 21st d after birth. In the F1 generation, the number of pups that decreased between the 1st and 21st d after birth was significantly higher in the middle dose group than in the control group; however, it was also significantly higher than in the other dosed groups. Furthermore, this difference was due to the values from only 2 dams, in one of which already birth did not proceed as usual. This finding was thus assessed as not due to the test substance.

No significant difference was observed in testis, epididymis, ovary (though the relative weights in the P generation were significantly lower in the lowest dose group than in the control group, those of the highest dose group were equal to those of the control group, so that an influence of the test substance on the organ weight was not assumed) and pituitary weight (absolute and relative weights were significantly higher in F1 generation females of the middle dose group than in the control group but no histopathological differences was observed between the high dose and control group). Histopathological examination of the reproductive organs did not yield findings that would reveal an influence of the test substance on the organs important for reproduction. Any deviations from the norm detected during the observations, autopsies and histopathological findings, with the exception of a significantly increased number of ovarian cysts in the control group of the P generation were about equally distributed among all 4 groups. Furthermore, in most cases, they were known as historically occurring changes and have no relation to the test substance.

Based on above, no adverse influence of the test substance (p-toluylenediamine sulphate) on female and male fertility of two generations of rats nor on the rearing of pups could be detected at any of the used dosages of 5, 15 and 45 mg/kg bw. The NOAEL for reproductive effects, as well as maternal toxicity was determined to be ≥45 mg/kg bw/day.

This two-generation reproduction toxicity study is classified as acceptable, and satisfies the guideline requirements of the OECD 416 method.
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
Justification for selection of Effect on fertility via oral route:
key study on 2,5- toulene diamine sulfate used for read-across (reliability 2)

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented publication, comparable to guideline with deviation
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Mice were used instead of rats (preferred species) and the high dose group did not fulfill the recommended number of at least 20 dams/group)
GLP compliance:
no
Limit test:
no
Test material information:
Composition 1
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Breeding Laboratories (Wilmington, MA)- Age at study initiation: 60-90 d- Weight at study initiation: Not reported- Housing: Prior to mating, the females were housed in polypropylene in a group of 10/cage and males were housed individually in another room in polypropylene mouse cages. The mated female mice with vaginal plugs were divided into experimental and control groups and were caged in a group of
Route of administration:
subcutaneous
Vehicle:
other: Sterile, distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in sterile, distilled water. VEHICLE:- Dose volume: 1% bw (10 mL/kg) The volume was based on the weight of each mouse on the day the test substance was administered.- Concentration of test material in vehicle: 1.6, 3.2, 4.8 and 6.4 mg/mL for 16, 32, 48 and 64 mg/kg bw respectively.- Lot/batch no: Gibco, Grand Island, NY; Lot # Not reported
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: Cohoused - M/F ratio per cage: 1:2- Length of cohabitation: Males and females were housed together, and the following morning the mice with vaginal plugs were caged (
Duration of treatment / exposure:
Animals were treated on Day 6-15 of gestation
Frequency of treatment:
Animals were treated once daily
Duration of test:
Animals were sacrificed on Day 18 of gestation
No. of animals per sex per dose:
The numbers of dams treated were as follows: 31 (control), 27 (16 mg/kg/day), 26 (32 mg/kg/day), 31 (48 mg/kg/day) and 11 (64 mg/kg/day).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels used in the study were chosen on the basis of toxicity information and were far in excess of any exposure levels likely for the average person, even an individual handling this substance.- Rationale for animal assignment: The dams were divided into control and experimental groups such that body weight differences between groups were minimized.- Rationale for route of administration: Due to the deficiencies inherent in dermal applications, it was decided that the subcutaneous route would closely mimic the absorption mechanism for hair dyes in humans, while insuring consistent, quantitative uptake of the test substance.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: Not reported- Cage side observations included: MortalityDETAILED CLINICAL OBSERVATIONS: NoBODY WEIGHT: Yes - Time schedule for examinations: Body weight gain was recorded for the time periods of Days 6-17POST-MORTEM EXAMINATIONS: Yes - Sacrifice on: On Day 18 of gestation (by cervical dislocation)- Organs examined: Reproductive status was determined with emphasis on uterus, uterine horns and general condition of each conceptus.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: YesExaminations included:- Gravid uterus weight: No- Number of corpora lutea: No- Number of implantations: Yes - Number of resorptions: Yes; The number of resorptions was recorded. However, the study did not specify if they were early or late resorptions.- Other: The general condition of each conceptus was recorded. The staining of implantation sites in the uteri of apparently non-pregnant females was achieved through use of ammonium sulfide.
Fetal examinations:
- External examinations: Yes, live fetuses were weighed individually and checked for external malformations. - Fetal sex ratio: Yes, the sex of each fetus was determined by internal (surgical incision below navel) examination.- Dead/Alive fetus: Yes - Number of stunted fetus: Yes, live fetuses weighting
Statistics:
The litter was considered as the experimental unit for analysis of data regarding embryotoxicity and teratogenicity. Statistical significance of differences between groups was determined by the Mann-Whitney U test, while Jonckheere’s test was employed to test the significance of dose response relationships. Two-tailed tests were performed and p<0.05 was selected as the level of statistical significance.
Details on maternal toxic effects:
Maternal toxic effects:yesDetails on maternal toxic effects:64 mg/kg/day was lethal to 9 of 11 pregnant mice. The 48 mg/kg/day dose was also toxic, as indicated by the deaths of 4 of 31 treated mice. Although none of the dose levels tested caused a significant reduction in weight gain during pregnancy, there was a significant trend (p<0.05) in that direction as the dose increased.
Dose descriptor:
NOAEL
Effect level:
32 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yesDetails on embryotoxic / teratogenic effects:Average fetal weight tended to decrease as the dose increased. There was a significant (p<0.05) decline in fetal weights at doses of 32, 48 and 64 mg/kg/day and therefore it was concluded that there was evidence of embryotoxicity. In contrast, there was no evidence of an effect on the percent of malformed fetuses, number of resorptions, number of fetal deaths and number of stunted fetuses at any dose group and thus the test substance was determined not to be teratogenic.
Dose descriptor:
NOAEL
Effect level:
>= 64 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
16 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Reproductive performance of 2,5-toluenediamine sulfate (2,5 TDS) in mice (Study # OP46771)

 

Dose Group (mg/kg/day)

Control

16

32

48

64

Number of dams receiving test material

31

27

26

31

11

Number of dams alive on Day 18

31

27

26

27

2

Number of dams pregnant*

24

25

20

23

2

Average weight gain (gm) during pregnancy (Days 6-17)

18.0

17.6

16.7

16.0

17.0

Total number of implants

295

324

253

270

30

Average number of implants'

12.3

13

12.7

11.7

15

Number of resorptions

28

38

19

20

3

Percent resorptions of total number of implants

9.5

11.7

7.5

7.4

10

Number of fetal deaths

7

4

9

4

0

Percent fetal deaths of total number of implants

2.4

1.2

3.6

1.6

0

Male/female-live fetuses

131/129

135/147

111/114

107/139

17/10

Number of stunted fetuses

2

0

0

6

0

Average number of live fetuses per dam

10.8

11.3

11.3

10.7

13.5

Average fetal weight** (g)

1.04±0.027

0.974±0.017

0.936±0.016

0.934±0.032

0.861±0.014

* = Included dams with resorptions

** = Dead and stunted fetus were excluded

 

Conclusions:
2 -methyl-p-phenylenediamine sulphate (2, 5-toluenediamine sulfate) when administered subcutaneously to mated female CD-1 mice at 0, 16, 32, 48and 64 mg/kg/day was considered non-teratogenic and non-emryo toxic at 64 and 16 mg/kg/day respectively. The NOAEL for maternal toxicity, teratogenicity and embryotoxicity were 32 , 64 and 16 mg/kg/day respectively.
Executive summary:

The teratogenicity study of 2 -methyl-p-phenylenediamine sulphate (2,5-toluenediamine sulfate) was determined following OECD guideline 414 (Prenatal Developmental Toxicity Study).

This study was designed to evaluate the teratogenic effects of 2 -methyl-p-phenylenediamine sulphate when administered subcutaneously to mated female CD-1 mice once daily from Day 6-15 of gestation at dose levels of 16, 32, 48 and 64 mg/kg/day. Control group (0 mg/kg bw) were administered with sterile, distilled water (vehicle) only.

 

Male and nulliparous female mice ( 60-90 days old) were obtained from Charles River Breeding Laboratories (Wilmington, MA). and were cohoused by placing two females into each male's cage for breeding . Vaginal plugs (Day 1 of gestation) were observed for the proof of pregnancy. By Day 6 of gestation, the dams were divided into experimental and control groups such that body weight differences between groups were minimized and were caged in a group of </=10 dams/cage.

The test solution was prepared in sterile, distilled water. The test solution and the control were administered once a day in a volume equivalent to 1% bw (10 mL/kg) on Days 6 -15 of gestation. The volume was based on the weight of each mouse on the day the test substance was administered at the following dose levels:

0 (31 dams), 16 (27 dams), 32 (26 dams), 48 (31 dams) and 64 (11 dams) mg/kg/day.

All animals were observed for mortality and body weight gain of the dams was recorded for the gestation periods of Days 6-17. On Day 18 of gestation the mice were killed by cervical dislocation and their reproductive status was determined. The number of resorptions was recorded. Implantation sites in each uterine horn were counted and the general condition of each conceptus was recorded. The staining of implantation sites in the uteri of apparently nonpregnant females was performed with ammonium sulfide.

Live fetuses were weighed individually, sexed and checked for external malformations. Number of stunted fetus (live fetuses weighing </=0.5 g or less than two thirds the mean of their larger littermates) were determined. At least one-third of the fetuses of each litter, as well as all stunted fetuses and those having external malformations, were examined for visceral alterations. The bodies of all fetuses were then processed for skeletal examination. The heads of those fetuses subjected to visceral examination (with the exception of any fetuses which had external head malformations) were cut off at the base and prepared for free-hand sectioning.

The litter was considered to be the experimental unit for analysis of data regarding embryotoxicity and teratogenicity. Statistical significance of differences between groups was determined by the Mann-Whitney U test, while Jonckheere's test was employed to test the significance of dose response relationships. Two-tailed tests were performed and p<0.05 was selected as the level of statistical significance.

In this study, treatment at 64 mg/kg/day resulted in mortality of 9 of 11 pregnant mice. The 48 mg/kg/day dose was also toxic, as indicated by the deaths of 4 of 31 treated mice. Although none of the dose levels tested caused a significant reduction in weight gain during pregnancy, there was a significant trend (p<0.05) in that direction as the dose was increased.

Average fetal weight tended to decrease as the dose was increased. A significant (p<0.05) decline in fetal weights at dosages of 32, 48 and 64 mg/kg/day was observed and therefore it was concluded that there was evidence of embryotoxicity. No evidence of an effect on the average percentage of malformed fetuses was observed at any of the dose levels and thus the test substance was determined not to be teratogenic.

Based on the results above, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity, teratogenicity and embryotoxicity were 32 , 64 and 16 mg/kg/day, respectively.

This teratogenicity study is classified as acceptable, and satisfies the guideline requirements of the OECD 414 method.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
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
Justification for selection of Effect on developmental toxicity: via oral route:
key study on 2,5- toulene diamine sulfate used for read-across (reliability 2)

Justification for classification or non-classification

Administration of p-Toluylenediamine sulphate by oral route in two generation study at 0, 5, 15 and 45 mg/kg bw/day was determined to have no adverse influence on female or male fertility and on rearing of pups in two generation at any of the dose levels tested.

2 -methyl-p-phenylenediamine sulphate (2, 5-toluenediamine sulfate) when administered subcutaneously to mated female CD-1 mice at 0, 16, 32, 48and 64 mg/kg/day was considered non-teratogenic and non-emryo toxic at 64 and 16 mg/kg/day respectively. The NOAEL for maternal toxicity, teratogenicity and embryotoxicity were 32 , 64 and 16 mg/kg/day respectively.

No classification was applied according to CLP criteria