Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on developmental toxicity

Description of key information

In conclusion, based on the results in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Adipic acid dihydrazide (ADH) was established as being 100 mg/kg, based on the unscheduled mortality of a female, the effects on body weights and food consumption in the maternal females and on body weights of male fetuses at treatment at 300 mg/kg.

Since lower (male) fetal weights were observed at the same dose level as the reduced body weight and food consumption in their dams, Adipic acid dihydrazide (ADH) need not to be classified as a developmental toxicant.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jan 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
pH: 9.5 at 10 g/L at 20 °Cwater solubility: 102 g/L at 20 °C
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Source: Charles River Deutschland, Sulzfeld, Germany - Age at study initiation: 10-14 weeks - Weight at study initiation: 171 - 271 g; per subgroup, the body weights were within ±20% of its mean per subgroup, except for one subgroup where they were within ± 30 % weight range. - Fasting period before study: no - Housing: individually housed in Macrolon plastic cages with sterilized sawdust as bedding material and paper as cage-enrichment/nesting material. - Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). - Water: free access to tap water - Acclimation period: at least 5 days prior to pairing DIT klopt toch niet, want ze zijn bij de supplier al gemate. ENVIRONMENTAL CONDITIONS - Temperature (°C): set to maintain 18-24 - Humidity (%): set to maintain 40-70 - Air changes (per hr): at least 10 - Photoperiod (hrs dark / hrs light):12/12 IN-LIFE DATES: From: 07 November 2016 To: 1 December 2016
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Formulations were placed on a magnetic stirrer during dosing and had the appearance of suspensions. No correction was made for the purity/composition of the test item. VEHICLE - Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch. - Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples taken from dose preparations, prepared on 14 November 2016 (day 6 of treatment), were analyzed according a validated method (ABL Project 16233).After sampling, all samples were stored and shipped on dry ice to the test site for formulation analysis, ABL B.V. WA Scholtenstraat 7, 9403 AJ, Assen, The Netherlands.Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 postcoitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
days 6 to 20 post-coitum, inclusive
Frequency of treatment:
once daily for 7 days per week
Duration of test:
necropsy on day 21 post-coitum
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of the dose range finder (Test Facility Study No. 513530). In the dose range finder females (n=6/dose) were given 100, 300 or 1000 mg/kg bw/d from days 6 to 20 post-coitum inclusive by oral gavage. Maternal findingsTwo (out of six) pregnant females at 1000 mg/kg and one (out of six) pregnant females at 300 mg/kg were sacrificed in extremis on day 19 post-coitum after showing signs of ill health, including lethargy, piloerection, hunched posture and/or pale faeces. No further mortality occurred in the 100 mg/kg treated group and vehicle controls during the study period. At 1000 mg/kg, piloerection and/or pale faeces with or without salivation were noted towards the end of treatment (from day17-18 post-coitum onwards) in all females (including the two early sacrificed females on day 19 post-coitum.No toxicologically significant clinical signs were noted up to 300 mg/kg, except for hunched posture, piloerection and lethargy in the early sacrificed female at 300 mg/kg on day 19 postcoitum.A dose related lower body weight gain was noted, achieving levels of statistical significance in females treated at 300 and 1000 mg/kg when compared to controls. On day 21 post-coitum, body weight gain after correction for the uterus over the treatment phase, i.e. day 6 to day 21, was also statistically significantly lower, in a dose related manner, in females at 300 and 1000 mg/kg in comparison with controls. A large variation in uterus weights was observed in females at 1000 mg/kg, with a mean of approximately half of that in the controls, i.e. 44.2 grams in high dose females compared to 81.7 grams in controls. The mean uterus weights of females at 100 and 300 mg/kg was considered similar to that in the other groups.Concurrent dose related decreases in food consumption were noted, more prominent during the first part of the treatment phase, achieving levels of statistical significance also in females treated at 300 and 1000 mg/kg when compared to controls.Reduced size of the thymus was observed in two early sacrificed females, in one of the two high dose females and in the mid dose female. Macroscopic examination of the femalessurviving until scheduled necropsy showed a reddish discolouration of the thymus in one female at 1000 mg/kg, which was considered to be related to treatment.No alterations were observed at macroscopic examination of the other high dose females and the females of the 100 and 300 mg/kg groups and vehicle controls.All females were found to be pregnant with viable fetuses, including the early sacrificed females.A dose related post-implantation loss was observed, all being early resorptions. The mean number of early resorptions noted were 0.5, 0.5 2.0 and 7.3 per litter for the control, 100, 300and 1000 mg/kg groups, respectively. The high(er) number of early resorptions were not equally distributed over the litters within the mid and high dose groups. In the mid dosegroup, 6 early resorptions were found in one litter and 0 – 2 in the other four litters. In the high dose group, two litters were found with 10 or 12 early resorptions and the two other with3 or 4 early resorptions.Fetal findingsLitter sizes were within normal limits for all groups.The male : female ratios were equal in litters of all groups.Fetal body weights were decreased for male and female fetuses at the 300 mg/kg dose group.External examination, including morphologically, of the fetuses did not show any treatment related abnormalities up to 1000 mg/kg.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: at least twice daily for mortality and viability DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy. The mated females that arrived on 08 November 2016, i.e. nos. 7-11, 28-33, 50-55 and 73-77, were not observed for clinical signs on Day 2 post-coitum, by mistake. It was suspected that these females were without any clinical signs, because of the absence of clinical signs in these females on subsequent days in the pretreatment phase (from Day 3 to Day 6 post-coitum). The omission of results on observations of these animals was therefore considered not to have affected the results of the study. BODY WEIGHT: Yes - Time schedule for examinations: days 2, 6, 9, 12, 15, 18 and 21 post-coitum FOOD CONSUMPTION: Yes - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected. POST-MORTEM EXAMINATIONS: Yes - Sacrifice on gestation day 21
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes Examinations included: - Gravid uterus weight: Yes - Number of corpora lutea: Yes - Number of implantations: Yes - Number and distribution of early resorptions: Yes - Number and distribution of late resorptions: Yes - Number and distribution of live and dead fetuses: yes - Weight and sex of each fetus: yes - Externally visible macroscopic fetal abnormalities: yes
Fetal examinations:
- External examinations: Yes, all per litter - Soft tissue examinations: Yes, half per litter - Skeletal examinations: Yes, half per litter - Head examinations: Yes, half per litter
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 group.• 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 Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral andskeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealedstatistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-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 calculatedfor 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 standarddeviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites)/number of corpora lutea) x 100Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/number of implantation sites) x 100Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs that were considered to be related to treatment were observed among females treated up to 300 mg/kg.Single or temporary observations of piloerection, hunched posture and/or chromodacryorrhoea were observed among a three high dose females treated at 300 mg/kg that survived until scheduled necropsy. In the absence of any corroborative finding in these animals they might have occurred by chance, despite the fact that these symptoms were comparable to those observed in the early sacrificed female. Therefore these observations were considered to be non-adverse and of no toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
One high dose female treated at 300 mg/kg was sacrificed in extremis on Day 17 post-coitum. Signs of ill health, comprising piloerection, hunched posture and chromodacryorrhoea, became apparent on day 13. Its health status gradually declined and marked body weight loss and reduced food consumption were noted over days 12 to 15 postcoitum. After a further body weight loss was seen on day 17 post-coitum (body weight of 161 grams on day 17 post-coitum) and pale faeces and emaciation were additionally observed, it was decided to sacrifice the animal.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was observed in high dose females from start of treatment onwards (day 6 post-coitum) until termination, achieving levels of statistical significance onall occasions during the treatment phase when compared to that in controls. Average absolute body weights on Day 21 post-coitum were 5% lower at 300 mg/kg compared to controls.On day 21 post-coitum, body weight gain over the treatment phase, i.e. day 6 to day 21, after correction for the uterus was also statistically significantly lower in high dose females in comparison with controls. The mean uterus weight of high dose females was similar to that in the other groups. The body weights and body weight gain after correction for the uterus in the low and mid dose groups remained in the same range as controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In females treated at 300 mg/kg, a lower food consumption was observed from start of treatment onwards (day 6 post-coitum) when compared to that in the other groups. Thelargest difference in food consumption was observed during the first part of treatment phase but gradually decreased during the progress of the study. Towards the end of the study, food consumption in high dose females was almost similar to that in the other groups, in particular the food consumption relative to body weight.The difference in food consumption between high dose females and controls reached levels of statistical significance over days 6-9, 9-12, 12-15 and 18-21 post-coitum for the absolute values and over days 6-9 and 9-12 for the relative values.Mean food consumption before or after correction for body weight for females treated at 30 and 100 mg/kg remained similar to the control level over the treatment period.
Water consumption and compound intake (if drinking water study):
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related macroscopic findings were observed in any of the animals of all dose groups.The emaciation of female no.72 and alopecia in female no.83 confirmed the symptoms observed in the in-life phase.The discolouration of the thymus and reddish foci in abdominal muscle tissue in a single female treated at 100 mg/kg was considered a fortuitous finding and of no toxicologicalsignificance.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
In females treated at 300 mg/kg, a relatively high mean percentage early resorptions per litter was observed (10.4 vs 3.8 in controls). This high mean value was caused by a high number of early resorptions in two (out of twenty) litters, as is also reflected by the large the standard deviation to the mean of this percentage of early resorptions. In these litters of females nos. 71 and 86, the percentage of early resorptions per litter was 82% and 64%, respectively, compared to maximal 17% early resorptions in the litters of the other high dose females.Litters with high post implantation losses are occasionally noted in rats of this age and strain.The presence of two such cases in the high dose group was considered to have occurred bychance and of no toxicological significance.The statistical significance noted for the higher mean value for pre-implantation loss infemales at 30 mg/kg when compared to controls, was considered to have occurred by chance.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
see description under pre- and post-implantation loss
Dead fetuses:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 100 mg/kg and one female at 300 mg/kg was not pregnant.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
mortality
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly lower mean male fetal body weight at 300 mg/kg was noted when compared to control. The mean male fetal body weights at 30 and 100 mg/kg and meanfemale fetal body weights in all treated groups remained in the same range as controls.Mean male fetal body weights were 5.5, 5.4, 5.4 and 5.1 grams for the vehicle control, 30, 100 and 300 mg/kg, respectively.Mean female fetal body weights were 5.2, 5.2, 5.2 and 5.1 grams for the vehicle control, 30, 100 and 300 mg/kg, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Mean sex ratios (males:females) were 55:45, 47:53, 52:48 and 53:47 for the vehicle control, 30, 100 and 300 mg/kg, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter sizes were 11.1, 10.7, 10.5 and 10.9 fetuses/litter for the vehicle control, 30, 100 and 300 mg/kg bw/day groups, respectively.The mean litter size of 10.9 fetuses/litter for the high dose group is excluding the litter of female no.72 (sacrificed in extremis on day 17 post-coitum) consisting of 10 (very small) livefetuses and one early resorption.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
An external malformation was only noted in one fetus at 30 mg/kg. This fetus showed inward rotation of both hind limbs without apparent skeletal origin.External variations were not seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg, four fetuses had an anomalous skeleton. The malformations were malpositioned metacarpals, severely malaligned sternebrae and bent limb bones. There were no skeletal malformations observed at lower dose levels, but due to the low incidences at 300 mg/kg and the fact that two malformations were already listed in historical control data (bent limb bones and severely malaligned sternebrae), all were considered of no toxicological relevance. Besides, there was a control fetus with bent limb bones and another one had a rib anomaly.Skeletal variations occurred at an incidence of 79.6%, 80.8%, 92.2% and 89.2% per litter in control, 30, 100 and 300 mg/kg group, respectively. The only noteworthy variation was the finding of 14th full rib(s) which occurred at an incidence of 11.1%, 12.0%, 17.7% and 20.0% of fetuses per litter in control, 30, 100 and 300 mg/kg group, respectively. The values at 100 and 300 mg/kg were above the historical control data range (0.0% - 13.1% per litter), but the concurrent control value was also high and statistical significance between dose groups was not reached. Therefore, and because the higher number of 14th full ribs at 100 and 300 mg/kg was not coincided with higher incidences for caudal shift of pelvic girdle (13.0%, 21.6%, 17.7% and 13.5% of fetuses per litter in control, 30, 100 and 300 mg/kg, respectively), it was considered that the presence of more fetuses with14th full ribs at 100 and 300 mg/kg occurred by chance and was not of any toxicological relevance.All other skeletal variations noted were considered not treatment related as they occurred in the absence of a dose-dependent relationship, infrequently and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The only viscerally malformed fetus in this study was a control fetus that had situs inversus which as such was considered a chance finding. Moreover, the three variations that occurred (small supernumerary lobes or appendix of the liver and convoluted ureters) occurred singly or at low incidences in the absence of a dose-related incidence trend andtherefore were not considered to be treatment related.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
fetal/pup body weight changes
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Results of analysis:

No test item was detected in the control formulation. The concentrations analyzed in the formulations of 30, 100 and 300 mg/kg were in agreement with

the target concentrations (i.e. mean accuracies between 90% and 110%).

The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Adipic acid dihydrazide (ADH) was established as being 100 mg/kg, based on the unscheduled mortality of a female, the effects on body weights and food consumption in the maternal females and on body weights of male fetuses at treatment at 300 mg/kg.Since lower (male) fetal weights were observed at the same dose level as the reduced body weight and food consumption in their dams, Adipic acid dihydrazide (ADH) need not to be classified as a developmental toxicant.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

Justification for classification or non-classification

Today there are no indications of an effect of the submitted substance on the reproduction or the developing fetus.

Additional information

Categories Display