Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Test proposal

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study planned
Study period:
After approval by ECHA
Justification for type of information:
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies: There are no GLP studies available covering reproductive toxicity information requirements.
- Available non-GLP studies: There are no non-GLP studies available covering reproductive toxicity information requirements.
- Historical human data: There are no historical human data available on reproductive toxicity for the substance.
- (Q)SAR: According to the ECHA Guidance in Information Requirements and Chemical Safety Assessment Chapter R 7a: Endpoint specific guidance, there are a large number of potential targets/mechanisms associated with reproductive toxicity which, on the basis of current knowledge, cannot normally be adequately covered by a battery of QSAR models.
- In vitro methods: According to the ECHA Guidance in Information Requirements and Chemical Safety Assessment Chapter R 7a: Endpoint specific guidance, The combination of assays in a tiered and/or battery approach may improve predictivity, but the in vivo situation remains more than the sum of the areas modelled by a series of in vitro assays. Therefore, a negative result predicting absence of a particular property for a substance with no supporting information cannot be interpreted as demonstrating the absence of a reproductive hazard with the same confidence as an animal study. A positive result predicting a particular reproductive hazard in a validated in vitro test could provide a justification for the need of further testing beyond the standard information requirement. However, because of limited confidence in this approach at this time, such a result in isolation would not be adequate to support hazard classification.
- Weight of evidence: There are no data available which are sufficient for a weight of evidence approach.
- Grouping and read-across: No substances or a category of substances are known which apply for read-across addressing reproductive toxicity.
- Substance-tailored exposure driven testing: not applicable
- Approaches in addition to above: not applicable
- Other reasons: not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The basic test design of an extended one-generation reproductive toxicity study (test method EU B.56./OECD TG 443 with Cohorts 1A and 1B, without extension of Cohort 1B to include a F2 generation, and without Cohorts 2A, 2B and 3) is a standard information requirement as laid down in column 1 of Section 8.7.3., Annex X. According to Column 2 Annex X of REACH Regulation an extended one-generation reproductive toxicity study does not need to be conducted if:
- the substance is known to be a genotoxic carcinogen and appropriate risk management measures are implemented, or
- the substance is known to be a germ cell mutagen and appropriate risk management measures are implemented, or
- the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure.
None of the points outlined above apply for the test substance. Thus, in order to fulfil information requirements stated in column 1 Annex X of REACH Regulation for substances manufactured or imported in quantities of 1000 tpa or more, an extended one-generation reproductive toxicity study (test method EU B.56./OECD TG 443 with Cohorts 1A and 1B, without extension of Cohort 1B, and without Cohorts 2A, 2B and 3 is proposed.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
The protocol as described in OECD guideline 443 with rats by the oral route is proposed.
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: According to OECD guideline 443
- Basis for dose level selection: According to OECD guideline 443. Available data from a subchronic repeated dose toxicity test (according to OECD 408) and a prenatal developmental toxicity study (according to OECD 414) will be considered.
- Inclusion/exclusion of extension of Cohort 1B: The study design needs to be expanded to include the extension of Cohort 1B to include a F2 generation if:
a) the substance has uses leading to significant exposure of consumers or professionals, taking into account, inter alia, consumer exposure from articles, and
b) any of the following conditions are met:
* the substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or
* there are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, or
* there are indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches.
Since the available data do not trigger the inclusion of extension of cohort 1B, only an extended one-generation reproductive toxicity study (test method EU B.56./OECD TG 443 with Cohorts 1A and 1B, without extension of Cohort 1B to include a F2 generation, and without Cohorts 2A, 2B and 3) is proposed.
- Termination time for F2: Not applicable: As an extension of Cohort 1B is not proposed, no F2 generation is included and the termination time does not need to be determined.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B and/or developmental immunotoxicity Cohort 3
The study design needs to be expanded to include the extension of Cohorts 2A/2B, and/or Cohort 3 if
* existing information on the substance itself derived from relevant available in vivo or non-animal approaches, or
* specific mechanisms/modes of action of the substance with an association to (developmental) neurotoxicity, or
* existing information from studies on effects caused by substances structurally analogous to the substance being studied suggesting such effects or mechanisms/modes of action.
Considering these points, the available data do not trigger an extension to include the extension of Cohort 1B, Cohorts 2A/2B, and/or Cohort 3. Furthermore, assessment of neurobehavior effects (functional observation battery and motor activity assessment) were included in the available OECD 408 study (Triskelion B.V., 2017). Treatment-related findings included sliding with the ventral parts of the head and neck over the bottom of the open field, dyspnoea, grunting respiration, sniffing, piloerection, salivation, serous nasal discharge, low arousal, soft and/or mucoid faces, diarrhoea, soiled perineum and soiled fur. No neurotoxic effects of treatment were observed from motor activity assessment in any of the dose groups during the 30-minute test period. Thus, the OECD 408 study did not indicate neurotoxic potential of the substance.
Therefore, an extended one-generation reproductive toxicity study (test method EU B.56./OECD TG 443 with Cohorts 1A and 1B, without extension of Cohort 1B, and without Cohorts 2A, 2B and 3) is proposed.
- Route of administration: The oral route is proposed because this is a possible route of human exposure. In addition, the dose levels are determined based on the available data from a subchronic repeated dose toxicity test (according to OECD 408) and a prenatal developmental toxicity study (according to OECD 414) in which the animals were also administered via the oral route.
Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

No adverse effects on development were found in a developmental toxicity study (OECD TG 414) with rabbits. Based on the results, the NOAEL for maternal toxicity and developmental toxicity were both determined to be ≥ 20 mg/kg body weight/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Feb 2017 to 21 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of the test material (as cited in study report): Zink bis(diethyldithiocarbamate)
- Batch No.: 60616004
- Date of receipt: 25 Jul 2016
- Expiration date: 14 Jul 2018
- Purity: ≥ 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient temperature

OTHER SPECIFICS:
- Appearance: White powder
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Eighty-eight time-mated female New Zealand White rabbits, were ordered from a colony maintained under SPF conditions at Centre Lago, Vonnas, France. The time-mated dams arrived at the test facility on gestation day (GD) 1 or 2.
- Age at study initiation: 4-5 months of age
- Weight at study initiation: 3.7-3.8 kg
- Housing: Animals were housed individually in type I rabbit cages (65.3 x 65.3 x 45 cm). The cages and bedding were changed at least weekly and cage enrichment was supplied.
- Diet: Feed was provided ad libitum from the arrival of the animals until the end of the study. The animals were fed a commercially available rabbit diet (Stanrab, SDS Special Diets Services, Witham, England) for nutrients and contaminants.
- Water: Drinking water was provided ad libitum from the arrival of the animals until the end of the study. The drinking water was given in polypropylene or glass bottles, which were cleaned weekly and filled up when necessary. Tap water for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied.
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21
- Humidity (%): 45- 65. The upper limit of relative humidity was higher than 70% for short periods of time, due to meteorological circumstances or because of wet cleaning activities.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
For each day of the study and for each dosing group the appropriate amount of test substance was weighed in a glass bottle. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred until visual homogeneity is obtained. All suspensions were continuously stirred on a magnetic stirrer during the dosing procedure, in order to maintain the homogeneity of the test substance in the vehicle.

VEHICLE
- Concentration in vehicle: 1-10 mg/mL
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no.: A1600985
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity and content of the test substance in the diet were conducted using ICP-MS.
- Stability: The presence of the test substance in the test dilutions was determined by analysis of the Zink content. Therefore it was not possible to determine the stability of the test substance under experimental conditions. Instead, fresh test dilutions were prepared daily.
- Homogeneity: The homogeneity (and content) of the test substance in the test dilutions were demonstrated in one batch, by analyzing three samples (taken at top-, mid- and bottom- of the vial) of each test dilution. Samples were taken in duplo.
Details on mating procedure:
Not relevant, as time-mated dams arrived at the test facility.
Duration of treatment / exposure:
23 consecutive days (gestation day 6 to 28)
Frequency of treatment:
Once daily
Dose / conc.:
2 mg/kg bw/day (actual dose received)
Remarks:
Low-dose
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Mid-dose
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
High-dose
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION
The dose levels for this prenatal development study were selected on the basis of the following considerations:
- In a prenatal developmental toxicity study with Ziram (CAS number 137-30-4) dose levels of 0, 3, 7.5 and 15 mg/kg bw/day Ziram (vehicle 1% Methylcellulose) were administered by oral gavage to mated New Zealand White rabbits from gestation day 7 up to and including gestation day 19 (n=16 animals /group). This resulted in mortality in the mid (one animal) and high dose group (one animal). In addition two animals (one in the control group and one in the mid dose group) died after showing a noisy respiration. In the high dose group maternal toxicity was observed, as evidenced by a reduced food intake and body weight gain. In addition developmental effects were observed in the high dose group, as evidenced by an increased post-implantation loss and a decreased mean fetal weight. At the mid dose level of 7.5 mg/kg bw/day maternal toxicity was observed (decreased body weight gain), but no embryofetal developmental effects.

- In a dose range finding study with the test substance dose levels of 5, 10 and 20 mg/kg body weight (vehicle corn oil, 2 mL/kg) were administered by oral gavage to mated New Zealand White rabbits from gestation day 6 up to and including gestation day 28 (n=4 animals/group). This resulted in no mortalities or morbidity other than 2/4 animals in the 5 mg/kg group showing (vaginal) blood loss on one day during gestation. A lower body weight gain and food consumption was observed in the 20 mg/kg group. This was not statistically significant due to the low number of animals (3 pregnant animals out of 4 animals per group). No significant effects were observed on the number and distribution of implantations in the uterus or fetus weight.

Based on the effects observed in the dose range finding study a dose level of 20 mg/kg body weight test substance was selected as high dose in the prenatal development study and is expected to induce maternal toxicity in terms of effects on food consumption and body weight gain. A low dose of 2 mg/kg test substance was anticipated to be a No Observed Adverse Effect Level (NOAEL) for maternal and developmental effects. The mid dose was set at 10 mg/kg body weight.
Maternal examinations:
CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded. Any animal showing signs of severe debility or intoxication, particularly if death appears imminent, were humanely killed to prevent loss of tissues by cannibalism or autolytic degeneration.

BODY WEIGHT:
The body weight of each animal was recorded on gestation days 3, 6, 9, 12, 15, 18, 21, 24 and 29. Body weights were be recorded at the time of discovery after intercurrent death, if feasible.

FOOD CONSUMPTION
Food consumption was measured per cage by weighing the feeders. The consumption was measured in the following intervals: GD3-6, GD6-9, GD9-12, GD12-15, GD15-18, GD18-21, GD21-24 and GD24-29.

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 29
- The dams were examined for gross abnormalities. Dams that were found dead or killed in extremis were also examined macroscopically and if present the number of corpora lutea and implantations were recorded. Maternal tissues showing severe macroscopic abnormalities were retained.
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 [If necessary the implantation sites were made visible following Salewski E. (1964)]
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of dead and live foetuses, number of grossly visible malformed foetuses and foetuses with external abnormalities, individual fetus weight and individual placenta weight of the live foetuses.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Other: The sex of the fetus was determined
Statistics:
Tests generally were performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**).
Statistical tests included in the decision tree for continues data included Shapiro-Wilks Test for normality, Levene’s Test Median adjusted, Anova, Kruskal Wallis, Dunnet or Turkey-Kramer and Dunn’s or Wicoxon’s Test.
Statistical tests included in the decision tree for dichotomous data included Chi-squared standard or Exact, Fisher Exact and Cochran-Armitage.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- All scarified animals or animals found dead showed damage to the esophagus or thorax upon macroscopic examination. Based on the macroscopic examination, the cause of death for these animals was considered to be related to technical dosing difficulties.
- One animal in the low dose group was sacrificed for humane reasons after the animal had gnawed and swallowed the gavage tube and showed dyspnea.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal in the control group delivered early on gestation day 28 and was sacrificed. One animal in the low dose group and two animals in the high dose group were found dead on days 11 and 26 of gestation, respectively.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight and mean body weight gain were comparable in all groups during treatment (See Tables 1 and 2 in ‘Any other information on results incl. tables’). All groups showed mean body weight loss at the start of dosing (from gestation day 6-9), that recovered thereafter. This was considered to the treatment (oral gavage) and vehicle (oil), but not related to the test substance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Mean food consumption was comparable in all groups.
- At the start of dosing, from gestation day 6-9, food consumption was statistically significantly higher in the mid dose group as compared to the control group. This was considered not related to treatment.
- Several animals randomly distributed over all groups showed decreased feed intake and were given Critical Care ® by oral gavage in order to stimulate bowel movement. As this consists of a watery substance and maximally 50 mL per day, this is not considered to have influenced the feed intake.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Based on the macroscopic observations in the esophagus and thorax, the cause of death Of the five animals that died or were sacrificed before scheduled necropsy was concluded to be related to the technical aspects of gavage-administration and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
no abortion sites were found
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- Pre-implantation loss (mean no. per animal/mean % per animal): 1.3/9.8; 1.6/14.3; 0.7/6.3; 1.6/12.6 (for the control, low dose, mid dose and high dose group, respectively).
- Post-implantation loss (mean no. per animal/mean % per animal): 0.8/7.8; 1.5/13.5; 0.8/7.4; 1.1/9.8 (for the control, low dose, mid dose and high dose group, respectively).
- The mean number of implantation sites was comparable in all groups (11.1, 9.9, 10.1, 10.4 for the control group, low dose, mid dose and high dose groups, respectively).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Number of dams with resorptions:
- control group: 6
- low dose: 6
- mid dose: 9
- high dose: 8
Early or late resorptions:
no effects observed
Description (incidence and severity):
The number and distribution of early and late resorptions was comparable in all groups. Mean pre-implantation loss and mean post-implantation loss were comparable in all groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
- The mean number of live fetuses was comparable in all groups (10.2, 8.4, 9.3 and 9.3 for the control group, low dose, mid dose and high dose groups, respectively).
- the total number of dams with dead fetuses was 3, 4, 1 and 2 for the control group, low dose, mid dose and high dose groups, respectively.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea was comparable in all groups (12.4, 10.7, 10.3 and 12.0 for the control group, low dose, mid dose and high dose group, respectively). Mean ovary weight was comparable in all groups. Mean uterus weight of the full and empty uterus were comparable in all groups (See Table 3 in ‘Any other information on results incl. tables’). Mean placenta weight was statistically significantly higher in the low dose group as compared to the control group. In absence of a dose-response relationship, this was considered not related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: absence of clinical signs and effects on body weight and feed intake in the high dose group
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetus weight was comparable in all groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See Table 4 in ‘any other information on results incl. tables’ for an overview of mean fetal weights
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
MALFORMATIONS
- Misshapen pinna of the ears was observed in one fetus in the control group.
- Umbilical hernia was observed in one fetus in the mid dose group.
- Agenesis of the head (visceral and skeletal tissue above the lower jaw) was observed in one fetus in the high dose group. This observation is known in the New Zealand White rabbits (see attachment for historical background data).
- A misshapen snout was observed in one fetus in the high dose group.
- Hyperflexion of the paws was observed in 5 fetuses from one litter in the control group, one fetus in the low dose group and two fetuses from the same litter in the high dose group.
- See Table 5 in ‘any other information on results incl. tables’ for the number and percentage of fetuses and litters

VARIATIONS
- External variations observed were related to fetus size, subcutaneous haemorrhages, pale skin and distended abdomen.
- See Table 5 in ‘any other information on results incl. tables’ for the number and percentage of fetuses and litters
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
MALFORMATIONS
- Fused ribs were observed in one animal in the control group and branched ribs were observed in another fetus from the same litter.
- One fetus in the low dose group showed multiple skeletal malformations besides external malformation (hyperflexion of the paws) and visceral malformation (agenesis of the gallbladder). Skeletal malformations observed in this fetus were: agenesis of the radius, metacarpals and several phalanges. The centrum of the 6th and 7th lumbar vertebra and the centrum of the 1st and second sacral vertebra were fused. In addition two ribs were fused and three or more sternebrae.
- Fused ribs were observed in one fetus in the low dose group and one fetus in the mid dose group and one fetus in the high dose group.
- Branched ribs were observed in two fetuses in the mid dose group. Three or more sternebra fused was observed in three fetuses in the mid dose group, and two fetuses in the high dose group. Fusion of the vertebral centrum was observed in one fetus in the high dose group.
- See Table 5 in ‘any other information on results incl. tables’ for the number and percentage of fetuses and litters

VARIATIONS
- Skeletal variations observed were mainly related to the ossification status of the bones and showed no treatment-related retardation in ossification.
- See Table 5 in ‘any other information on results incl. tables’ for the number and percentage of fetuses and litters
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Malformations: A hernia of the diaphragm was observed in one fetus in the control group. Agenesis of the gallbladder was observed in one fetus in the low dose group.
- Variations: Visceral variations were observed in brain, liver, heart and great vessels, ureters, urinary bladder and gallbladder and female reproductive organs (uterus and ovaries).
- See Table 5 in ‘any other information on results incl. tables’ for the number and percentage of fetuses and litters
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Mean placenta weight was statistically significantly higher in the low dose group as compared to the control group. In absence of a dose-response relationship, this was considered not related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of effects on the mean number of implantation sites, early and late resorptions, live fetuses, fetus weight and fetal external, visceral and skeletal observations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

ANALYTICAL VERIFICATION OF DOSES OR CONCENTRATIONS

- Linearity: The correlation coefficient was 1.000 during the run which was performed during this study and therefore the preset criterion (the correlation coefficient of the calibration curves should be ≥ 0.996) was met.

- Homogeneity: The RSD between the mean concentrations at three different locations was < 5% for all dose levels and/or p was ≥ 0.01. Therefore the test substance was considered to be homogeneously distributed in the gavage liquids.

- Content: The concentration of the test substance was close to intended (90-110%) for all gavage liquids at all dose levels.

Table 1: Mean Body weights, Sex: Female - Phase:Gestation

 

Control

0 mg/kg

Low dose

2 mg/kg

Mid dose

10 mg/kg

High dose

20 mg/kg

Day 3

3,818.650a

3,779.390

3,716.810

3,774.379

Day 6

3,886.365a

3,854.848

3,845.548

3,857.400

Day 9

3,842.045a

3,811.262

3,832.848

3,810.121

Day 12

3,909.220a

3,875.900

3,877.729

3,874.074

Day 15

4,001.930a

3,953.295

3,958.871

3,958.053

Day 29

4,115.653k

4,042.932

3,992.438

4,103.117

Statistic Profile = DecisionTree, * = p < 0.05, ** = p < 0.01, X = Group excluded from statistics

a=ANOVA; k=KRUSKAL-WALLIS

 

Table 2: Mean Body weight change, Sex: Female - Phase:Gestation

 

Control

0 mg/kg

Low dose

2 mg/kg

Mid dose

10 mg/kg

High dose

20 mg/kg

Day3 -> 6

67.715u

75.457

128.738*

83.021

Day6 -> 9

-44.320k

-43.586

-12.700

-47.279

Day9 -> 12

67.175a

64.638

44.881

63.953

Day12 -> 15

92.710a

77.395

81.143

83.979

Day15 -> 29

105.016k

90.516

33.567

145.822

Day3 -> 29

293.063k

288.674

275.629

341.017

Statistic Profile = DecisionTree, * = p < 0.05, ** = p < 0.01,

d = day; u=KRUSKAL-WALLIS-DUNN; k=KRUSKAL-WALLIS; a=ANOVA

 

Table 3: Mean Uterine Weight, Sex:Female - Phase:Gestation, Day 29

 

Control

0 mg/kg

Low dose

2 mg/kg

Mid dose

10 mg/kg

High dose

20 mg/kg

Uterine Weight [g]

487.057a

436.638

443.154

460.763

Empty Uterine Weight [g]

54.8007a

52.7177

51.1444

57.1458

Statistic Profile = DecisionTree, * = p < 0.05, ** = p < 0.01

a=ANOVA

Table 4: mean fetal weights

 

Control

0 mg/kg

Low dose

2 mg/kg

Mid dose

10 mg/kg

High dose

20 mg/kg

Fetus Weight (g)

33.6a

33.9

32.9

33.3

Fetus Weight of Male Fetuses (g)

33.8a

35.4

33.4

33.5

Fetus Weight of Female Fetuses (g)

33.2a

33.2

32.2

33.1

Statistic Profile = DecisionTree, * = p < 0.05, ** = p < 0.01

u=KRUSKAL-WALLIS-DUNN; k=KRUSKAL-WALLIS; a=ANOVA

Table 5: Total fetal observations

 

 

Control

0 mg/kg

Low dose

2 mg/kg

Mid dose

10 mg/kg

High dose

20 mg/kg

Fetuses Examined (N)

 

194

160

196

168

Litters evaluated (N)

 

19

19

21

18

Total M - Malformation

Litters Affected (N)

4cx

2

5

7

 

Litters Affected (%)

21.1

10.5

23.8

38.9

 

Fetuses Affected (N)

9

2

5

8

 

% per Litter (Mean)

6.6k

3.4

2.5

4.9

Total V - Variation

Litters Affected (N)

19cx

19

21

18

 

Litters Affected (%)

100.0

100.0

100.0

100.0

 

Fetuses Affected (N)

161

153

178

155

 

% per Litter (Mean)

81.8u

95.8*

91.4

91.8

Statistic Profile = DecisionTree + FisherExact, * = p < 0.05, ** = p < 0.01

cx=CHI-SQUARE-EXACT, k=KRUSKAL-WALLIS, u=KRUSKAL-WALLIS-DUNN

Conclusions:
Based on the results, the NOAEL for maternal toxicity and developmental toxicity were both determined to be ≥ 20 mg/kg body weight/day.
Executive summary:

In a GLP-compliant developmental toxicity study performed according to OECD guideline 414, the test substance was evaluated for its prenatal developmental toxicity in pregnant female New Zealand White rabbits (Triskelion B.V., 2017). The test substance was administered daily by oral gavage as an suspension in corn oil to groups of 22 mated females in doses of 0 (control), 2, 10 and 20 mg/kg bw on gestation days (GD) 6 through 28. A dose volume of 2 mL/kg body weight was applied in all groups and corn oil was used as vehicle. In-life parameters included signs of morbidity and mortality, body weight, food consumption. On gestation day 29 the dams were sacrificed and examined macroscopically. Foetuses, placentas and reproductive organs were weighed. The foetuses were macroscopically examined and processed for visceral and skeletal examinations. The content and homogeneity of the test substance in the vehicle were confirmed by analysis.

With regard to maternal toxicity, no test substance related deaths or clinical signs were observed. Five unscheduled deaths were related to technical difficulties in gavage administration and not considered related to the test substance. No treatment related effects on body weight and food consumption were observed. No effects were observed on the mean number of corpora lutea, implantation sites and the mean number of live foetuses.

As to reproductive performance, Out of 22 mated females per group, 19, 19, 21 and 18 (for the control group, low, mid and high dose group, respectively) animals survived and were pregnant at necropsy. Mean pre-implantation loss and post-implantation loss was comparable in all groups.

After foetal examination, no differences were observed in foetal sex and weight or in placental weight. External, visceral and skeletal examination of the foetuses showed a limited number of malformations and variations in several organs and bones in all groups, including the control group. Based on the incidence and distribution of the external, visceral and skeletal malformations and variations observed, no treatment-related effects were observed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP compliant OECD 414 study
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

In a GLP-compliant developmental toxicity study performed according to OECD guideline 414, the test substance was evaluated for its prenatal developmental toxicity in pregnant female New Zealand White rabbits (Triskelion B.V., 2017). The test substance was administered daily by oral gavage as an suspension in corn oil to groups of 22 mated females in doses of 0 (control), 2, 10 and 20 mg/kg bw on gestation days (GD) 6 through 28. A dose volume of 2 mL/kg body weight was applied in all groups and corn oil was used as vehicle. In-life parameters included signs of morbidity and mortality, body weight, food consumption. On gestation day 29 the dams were sacrificed and examined macroscopically. Foetuses, placentas and reproductive organs were weighed. The foetuses were macroscopically examined and processed for visceral and skeletal examinations. The content and homogeneity of the test substance in the vehicle were confirmed by analysis.

With regard to maternal toxicity, no test substance related deaths or clinical signs were observed. Five unscheduled deaths were related to technical difficulties in gavage administration and not considered related to the test substance. No treatment related effects on body weight and food consumption were observed. No effects were observed on the mean number of corpora lutea, implantation sites and the mean number of live foetuses.

As to reproductive performance, Out of 22 mated females per group, 19, 19, 21 and 18 (for the control group, low, mid and high dose group, respectively) animals survived and were pregnant at necropsy. Mean pre-implantation loss and post-implantation loss was comparable in all groups.

After foetal examination, no differences were observed in foetal sex and weight or in placental weight. External, visceral and skeletal examination of the foetuses showed a limited number of malformations and variations in several organs and bones in all groups, including the control group. Based on the incidence and distribution of the external, visceral and skeletal malformations and variations observed, no treatment-related effects were observed.

Based on the results, the NOAEL for maternal toxicity and developmental toxicity were both determined to be ≥ 20 mg/kg body weight/day.

 

Developmental toxicity of the test substance was also studied by Nakaura et al. (1984). The test substance was administered at dose levels of 31.25, 64.2, 125 and 250 mg/kg bw/day as suspension in olive oil to groups of pregnant Wistar rats (21-23 animals/group) during days 7 to 15 of gestation. On gestation day 20, 14 rats from the control and high dose groups and 15 rats from the other test groups were opened under anaesthesia to inspect the uterus, number of corpora lutea, number of implants, sex ratio and number of live and dead foetuses. The other rats from each group were allowed to give natural birth, and post-natal development of the pups was examined. The assessed parameters were number of pups, mortality rate, outward abnormalities, skeletal and soft tissue abnormalities and body weight, as well as ear auricle extension, tooth bud collapse or emergence, fur emergence, eyelid opening and timing for testes descent and vagina opening. Pups were allowed to wean and the observation continued till age 10 weeks, after which animals were sacrificed and gross pathological and organ weight examinations were performed. No signs of maternal toxicity were noted in the controls and test groups of 31.25 and 62.5 mg/kg bw/day. In the 125 mg/kg group, while no change in the average weight trend was seen, minor cases of diarrhoea were observed in 5 rats out of 22 rats from the 6th day after start of administration (gestation day 12) through the 8th day (gestation day 14). In the 250 mg/kg group, minor suppression of body weight increase was seen from the 2nd day after start of administration (gestation day 8), and in all cases piloerection, diarrhoea, bleeding around the eyes, and debilitation were observed, with 7 rats out of 21 dying between gestation day 9 and day 13. The pregnant rats that avoided death continued to show minor suppression of body weight increase even after administration was ended. Therefore NOAEL for maternal toxicity was set at 125 mg/kg bw/day, while the dose level of 62.5 mg/kg bw/day is considered to be a NOEL. No significant differences were found in the number of corpora lutea, implantations sites, implantation rates, live and dead foetuses, sex ratio and foetus weights between the controls and the test groups. In the external abnormality test, no abnormal foetuses were observed in the control group and in the treated groups of 125 mg/kg or less. In the 250 mg/kg group, one case of a foetus with a cleft palate was found. However, this occurrence rate was 0.6%, and was not a significant difference when compared with the control group. In the internal organs test, no abnormal foetuses were observed among the surviving foetuses. Abnormalities thought to be skeletal malformations were not observed in the control group and in the exposed groups of 125 mg/kg or less. In the 250 mg/kg group, one case of a foetus with a cleft palate was found (0.8%). However, this occurrence frequency of skeletal malformation foetuses was low, and was not a significant difference when compared with the control group. Abnormalities that could be considered skeletal deformations were observed in all groups, including the control group. Cervical ribs were observed in 1.5 to 8.9% of all groups. Foetuses with shortened or split cervical arches were observed in 1.7% of the 62.5 mg/kg group and 4.2% of the 250 mg/kg group. Deformations (vestigial conditions, dual sphere conditions) of the thoracic centra were observed in 3.0 to 11.0% of all groups, split thoracic centra was observed in 2.7% of the control group, 1.6% of the 31.25 mg/kg group, 0.7% of the 62.5 mg/kg group, and 2.2% of the 250 mg/kg group. Foetuses with sternebrae abnormalities (deformation, splitting, fusion, deficiency) included 64.0% of the control group, 59.7% of the 31.25 mg/kg group, 63.6% of the 62.5 mg/kg group, 64.1% of the 125 mg/kg group, and 81.4% of the 250 mg/kg group. Lumbar ribs were observed in 31.1 to 58.5% of all groups, including the control group. Shortened pubic bones were observed in 0.8% of the 31.25 mg/kg group. Nevertheless, the occurrence rates for these skeletal deformations did not show significant differences between the control group and the dosed groups. For the ossification state, the bone number for the metacarpal bone, metatarsal bone, and sacro-cardal vertebrae was determined. In every case, there was no significant difference in bone number between the target group and the dosed groups. No significant differences in body weight were observed between the test groups and control groups up till the age of 10 weeks, when the study was terminated. For the ear auricle extension, tooth bud collapse or emergence, fur emergence, eyelid opening, and timing for testes descent and vagina opening of newborn pups, each measurement period showed no significant difference between the control group and the dosed groups. Therefore a NOAEL for developmental toxicity was set at the highest tested dose of 250 mg/kg bw/day.

Justification for classification or non-classification

Based on the absence of adverse effects in the developmental studies in rabbtis and rats, classification is not warranted for developmental toxicity in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.