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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
α,α'-propylenedinitrilodi-o-cresol
EC Number:
202-374-2
EC Name:
α,α'-propylenedinitrilodi-o-cresol
Cas Number:
94-91-7
Molecular formula:
C17H18N2O2
IUPAC Name:
α,α'-propylenedinitrilodi-o-cresol
Details on test material:
- Test Item: Alpha,alpha'-Propylenedinitrilodi-o-cresol
- BASF Test Item No.: 11/0600-1
- Batch Number: 11000129U0
- Purity: >99 corr. area %
- Expiration Date: February 04, 2013
- Physical state, appearance: Liquid, highly viscous, yellowish
- Storage conditions: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: animals of comparable size and weight
- Housing: 5 animals/sex and cage pre-mating, one male and one female during mating, females individually after mating, males group-housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was chosen based on trial formulations performed at WIL Research Europe
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. 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 occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
daily
Details on study schedule:
not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day dose range finding study.
- Rationale for animal assignment (if not random): by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS (for mortality) Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were made in all animals, at least immediately after dosing (on the peak period of anticipated effects after dosing). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were conducted at least immediately after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

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

OTHER:
Further details on repeated dose parameters are given in the respective repeated dose toxicity section of this IUCLID.
Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
Parameters examined in [P] male parental generation:
[testis weight, epididymis weight]
From the selected 5 males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
Litter observations:
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made for all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex
Sex was determined for all pups on Days 1 and 4 of lactation.

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-6 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
All animals were subjected to macroscopic examination 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.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs will be collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), (Skin), Spinal cord -cervical, midthoracic, lumbar, (Male and female mammary gland area), Spleen, Femur including joint Sternum with bone marrow, Heart, Stomach, Ileum, Testes, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination [Examination of the mammary gland area was required for females nos. 71 and 76 with total litter loss.].

HISTOPATHOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

From the selected 5 males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

A peer review on the histopathology data was performed by a second pathologist.

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously.
- All gross lesions of all animals (all dose groups).
- Stomach the selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in Group 4.
- Thymus of the selected 5 animals of Groups 2 and 3 (females), based on (possible) treatment-related changes in this organ in Group 4.
- The reproductive organs of all animals of Groups 1 and 4, and of males 31 and 36 (Group 4) and females 71 and 763 (Group 4); both females had a total litter loss.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid
Postmortem examinations (offspring):
Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-6 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
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 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis non-parametric ANOVA test to determine intergroup differences followed by the Wilcoxon test 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 lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), Duration of gestation
Offspring viability indices:
For each group, the following calculations were performed: Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.

Salivation seen after dosing among animals of the 25, 75 and 250 mg/kg bw/day dose group was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.

Incidental findings that were noted included regurgitation together with rales at a slight degree, chromodacryorrhoea (snout) and scabbing on different parts of the body. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.No clinical signs were noted for the Group 3 female.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no mortalities that were related to the test substance. One female at 75 mg/kg bw/day and one female at 250 mg/kg bw/day were found dead after dosing on Day 9 of the pre-mating period. Before death, the Group 4 female showed severe restlessness together with breathing difficulties (laboured respiration and gasping).

Gross findings at necropsy included perforation of the oesophagus (granulation tissue and an inflammatory process at microscopic examination), discoloration of the lungs (reddish or dark-red) and Harderian glands (pale) and/or (yellowish) watery-cloudy fluid in the thoracic cavity for both females. These findings were suggestive of a gavage accident as cause of death. In this context it is noteworthy that animals treated with the test substance (Groups 2-4) were more restless during dosing than controls.

Two females at 250 mg/kg bw/day had to be euthanized after total litter loss on Day 1 of lactation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and food consumption were within the normal range. No toxicologically relevant changes in body weight and body weight gain were noted.

Body weights and body weight gains were statistically significantly lower in males at 75 and 250 mg/kg bw/day on Day 8 of the pre-mating period and thereafter. However, the differences to controls were slight, and values remained within the range considered normal for rats of this age and strain (5-95% confidence interval body weight gain on mating Day 15: 11-30%). Therefore, these differences were considered not to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Body weights and food consumption were within the normal range.

No toxicologically relevant changes in food consumption before or after correction for body weight were noted.

The slightly, but statistically significantly lower food intake (absolute and/or relative to body weight) noted for females at 75 mg/kg bw/day from Days 7-11 post-coitum and for females at 250 mg/kg bw/day from Days 0-4 and 7-11 post-coitum was considered to be of no toxicological relevance, as values remained within the range considered normal for rats of this age and strain and/or changes occurred in the absence of a treatment-related distribution.

At the individual level, both absolute and relative food intake was lower for three females at 250 mg/kg bw/day (nos. 73, 75 and 77) during lactatio. This was at least for females 73 and 75 in part due to the relatively small litter size (5 and 4 pups, respectively).
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The following treatment-related microscopic findings were noted:

Stomach (both sexes)
- An increased incidence and severity of hyperplasia of the squamous epithelium with hyperkeratosis was recorded at 250 mg kg bw/day in 6/6 males (moderate) and in 5/5 terminal females (2: minimal, 2: slight, 1: moderate).
- A lymphogranulocytic inflammation of the forestomach at a minimal degree was recorded at 250 mg/kg bw/day in 6/6 males and 2/5 terminal females.

Thymus (females)
- A slightly increased incidence and severity of lymphoid atrophy was recorded in 3/5 terminal females (1: minimal, 2: slight) at 250 mg/kg bw/day.
Minimal lymphoid atrophy of the thymus was also recorded in a single female of each the control and 75 mg/kg bw/day group and a single male at 250 mg/kg bw/day.

The minimal lymphoid atrophy of the thymus in single animals and all remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain.

Examination of the reproductive organs of the animals (incl. assessment of the integrity of the spermatogenetic cycle for males) did not reveal any abnormalities up to 250 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Two females at 250 mg/kg bw/day had a total litter loss on Day 1 of lactation, resulting in a gestation index of only 77.8% for Group 4 as compared to 100% for the remaining groups. The reason for the total litter loss could not be established as part of this study. Based on the in-life data there were no indications for a poor condition of these two females, and examination of the reproductive organs of the animals that failed to deliver healthy offspring (male 31, 36, female 71, 76) did not reveal any abnormalities.

No treatment-related toxicologically significant changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

There was a trend towards slightly lower numbers of corpora lutea and implantation sites at 250 mg/kg bw/day. This was mainly attributable to females 75 and 79 that had 7 corpora lutea each and 5 and 7 implantation sites, respectively. Since lower numbers were also seen for control female 42 (8 corpora lutea and 8 implantation sites), this finding was considered of no toxicological relevance.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: total litter loss in two females leading to a decreased gestation index

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental clinical symptoms of pups consisted of no milk in the stomach, missing tail apex, and wound and scabbing on the head. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and they were therefore considered to be of no toxicological relevance.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of living pups at first litter check was significantly lower at 250 mg/kg bw/day compared to controls. A total of 15 dead pups were recorded for 4 litters in the high dose group compared to 1 pup/1 litter, 2 pups/1 litter and 0 pups at 0, 25 and 75 mg/kg bw/day, respectively. Two out of these four females at 250 mg/kg bw/day (nos. 71 and 76) had a total litter loss on Day 1 of lactation with 11 and 1 dead pup, respectively.

There were no treatment-related effects at the lower dose levels of 25 and 75 mg/kg bw/day.

One control pup went missing on Day 3 of lactation. It was most likely cannibalised.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered to have been unaffected by treatment. All values remained within the normal range of biological variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental macroscopic findings of pups that were found dead included beginning autolysis and/or no milk in the stomach. The only macroscopic finding among surviving pups was a missing tail apex for one control pup. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mean live litter size was slightly lower in the high dose group when excluding the two dams with total litter loss

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Total litter loss was noted for two dams at 250 mg/kg bw/day at first litter check, leading to a decreased gestation index of 77.8%. In addition, excluding these two dams, mean live litter size was slightly lower in this group (control: 11.0; 25 mg/kg bw/day: 10.2; 75 mg/kg bw/day: 12.4; 250 mg/kg bw/day: 8.3). No effects on the number of live and dead pups were observed at the lower dose levels.

 

There were no adverse effects on the duration of pregnancy, parturition, maternal care, sex ratio of the pups and pup development after first litter check regarding clinical signs, body weight and macroscopy up to 250 mg/kg bw/day.

Gestation

Gestation index was decreased at 250 mg/kg bw/day, but 100% for the remaining groups, including controls. Duration of gestation was comparable for all groups.

 

Parturition/maternal care

No signs of difficult or prolonged parturition were noted among the pregnant females.

 

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

No deficiencies in maternal care were observed.

 

Early postnatal pup development

A higher mortality was noted in the 250 mg/kg bw/day group, but not at the lower doses of 25 and 75 mg/kg bw/day. For further details, see sectionMortality.

 

Postnatal loss, viability index, sex ratio, clinical signs, body weight, and external macroscopy did not reveal any treatment-related findings up to 250 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
Parental results
Repeated administration of alpha,alpha’-Propylenedinitrilodi-o-cresol to male and female Wistar Han rats caused salivation after dosing, which was transient and occurred at a very low to low incidence at 25 and 75 mg/kg bw/day, and at a higher incidence at 250 mg/kg bw/day. This finding was considered to be probablya physiological response and therefore not as adverse.
 
An increased incidence and severity of hyperplasia of the squamous epithelium with hyperkeratosis (up to a moderate degree) and lymphogranulocytic inflammation of the forestomach (minimal degree) were recorded at 250 mg kg bw/day (both sexes). This finding is suggestive for an irritant potential of the test substance. In line with this, macroscopic abnormalities indicative for irritation of the mucosa of the stomach were noted at 300 and 800 mg/kg bw/day (both sexes) in the previous dose range finding study.
 
The trend towards lower thymus organ weights (absolute and relative to body weight) noted at 250 mg/kg bw/day (both sexes) together with a slightly increased incidence and severity of lymphoid atrophy (up to a slight degree; females only) was not considered as an adverse effect, since changes were relatively slight. They were most likely secondary to stress caused by the local effects of the test substance on the stomach.
 
No adverse effects were noted in any of the remaining parental parameters investigated in this study (i.e. mortality, clinical appearance, functional observations (incl. locomotor activity), body weight, food consumption, clinical laboratory investigations (haematology and clinical biochemistry), and macroscopic examination).
 
Reproductive results
Two females at 250 mg/kg bw/day had a total litter loss on Day 1 of lactation, resulting in a gestation index of only 77.8% for Group 4 as compared to 100% for the remaining groups. The reason for the total litter loss could not be established as part of this study. Based on the in-life data there were no indications for a poor condition of these two females, and examination of thereproductive organs of the animals that failed to deliver healthy offspring (male 31, 36, female 71, 76) did not reveal any abnormalities.
 
No treatment-related toxicologically significant changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).
 
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
 
Developmental results
Total litter loss was noted for two dams at 250 mg/kg bw/day at first litter check, leading to a decreased gestation index of 77.8%. In addition, excluding these two dams, mean live litter size was slightly lower in this group.
 
No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e.duration of pregnancy, parturition, maternal care, sex ratio of the pups, and earlypostnatalpup development consisting of mortality, clinical signs, body weight and macroscopy).
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 250 mg/kg bw/day for systemic toxicity, 75 mg/kg bw/day based on local irritation at the stomach;
Reproduction NOAEL: 75 mg/kg bw/day;
Developmental NOAEL: 75 mg/kg bw/day.