Diallyl hexahydrophthalate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2018 to 03 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

These deviations are considered to have no impact on the outcome of the study or on interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals ; 7 days
- Basis for dose level selection ; The dose levels were selected by the Study Director in consultation with the Sponsor based on available acute oral toxicity data (LD50 between 300 and 2000 mg/kg bw in rats [3]) and information from a Dose Range Finding study in the rat (Citoxlab study code 17/277-220PE [4]) where test item related mortality was seen at 1000 mg/kg bw and slight toxicity was seen at 300 mg/kg bw/day.
- Inclusion/exclusion of extension of Cohort 1B ; not examined
- Termination time for F2 ; only F1
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B ; no
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 ; no
- Route of administration ; oral gavage
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Osaka Soda; Bx 40201
- Expiration date of the lot/batch:
26 January 2019
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room Temperature, protected from light
- Stability under test conditions:
no data
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
none
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/277-220PE [4]).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Commercial laboratory animal supplier.
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
13 weeks
- Weight at study initiation:
Males: 429 – 494 g, females: 255 – 313 g
- Fasting period before study:
- Housing:
Type II polycarbonate cage
- Diet:
ssniff® SM R/M "Autoclavable complete diet for rats and mice ad libitum
- Water: ad libitum
- Acclimation period:
12 days

DETAILS OF FOOD AND WATER QUALITY:
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 883 29966, expiry date: 31 October 2018 and batch number: 840 33675, expiry date: 31 January 2019), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19.0 – 25.0 °C (target range 22 ± 3 °C)
- Humidity (%):
34 – 69 % (target range 30-70 %)
- Air changes (per hr):
15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light):
12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 03 July 2018 To: 07 August 2018

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
Formulations were prepared up to 4 days before use and were kept at room temperature until use.
- Mixing appropriate amounts with (Type of food):

- Storage temperature of food:
room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Based on the results of a short solubility test performed at the Test Facility, PEG 400 was selected as vehicle for this study in agreement with the Sponsor, based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study [4].
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL / kg bw
- Lot/batch no. (if required): A0378559
- Purity:

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 7 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed using an HPLC-UV method in the Analytical Laboratory of Citoxlab Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations three times during the study (around the beginning and end, and approximately midway during the treatment), one set to analyse (which were collected in replicates) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control (vehicle)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Mid dose

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Study Director in consultation with the Sponsor based on available acute oral toxicity data (LD50 between 300 and 2000 mg/kg bw in rats [3]) and information from a Dose Range Finding study in the rat (Citoxlab study code 17/277-220PE [4]) where test item related mortality was seen at 1000 mg/kg bw and slight toxicity was seen at 300 mg/kg bw/day.
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
yes
- Rationale for selecting satellite groups:
n/a
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OTHER:

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

Additionally, vaginal smears were prepared and examined for each surviving female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia]

GROSS EXAMINATION OF DEAD PUPS:
[no, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Maternal animals: All surviving animals were dsed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
All F1 offspring were terminated on Day 13 post-partum (F1 offspring selected for blood sampling on PND4 were terminated on that day). In order to allow for overnight fasting of dams with urine collection on PPD14, the offspring were euthanized on PPD/PND13 and the dams on PPD/PND14.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Reproductive indices:

The calculations were generally made for surviving animals and therefore they are not applicable for groups with unscheduled deaths. The mating indices were 100% in all groups, except the High dose group where three males died before a successful mating could happen. The female fertility indices were 100% in the Control, Low and Mid dose groups and 75% in the High dose group (3 non-pregnant females out of the 12 animals).

Offspring viability indices:

The number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values in the Low and Mid dose groups. Overall, there were no treatment-related effects on pup mortality and on the viability of pups on PND0, 4 and 13 in these groups.

In the High dose group two females (#4503, 4507) had high number of stillborns and their few live born pups also died within a few days. Animal #4503 died after parturition because of massive hepatocellular necrosis. Animal #4507 survived the study but also massive hepatocellular necrosis was seen at histopathology. Because of this, it is considered that the high number of stillborns in these litters were attributed to a secondary effect of maternal toxicity. Of the 5 High dose females that survived, one had massive hepatocellular necrosis, must pups were born dead and the 3 living pups died in the first 4 days of lactation; the other 4 dams in the High dose group had normal litters and their pups had normal survival indices (at or close to 100%).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the Low and Mid dose groups during the study. The incidental piloerection in one Mid dose male (#3004) on Day 17 was considered as not test item-related.

In the found dead or pre-terminally euthanized High dose males, liquid faeces (4/4), hunched back (4/4), piloerection (4/4), slight or moderate decreased activity (3/4), red discharge at the nose or snout (3/4), noisy respiration (1/4) and red faeces (1/4) were observed. Similar findings were seen in one animal from Day 5, but it completely recovered from Day 20. Besides these, hunched back, piloerection or noisy respiration were occasionally seen in the other surviving animals for 1-2 days. Three High dose males were completely symptom-free during the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One High dose male was found dead on Day 17 and three High dose males were pre-terminally euthanized between Day 14 and 20. This was around the mating period, therefore three animals had to be replaced with other males to mate the females and one male was preterminally euthanized after successful mating.

In the High dose female group, four animals were found dead around the end of the gestation period or in the beginning of lactation period:

• : Animal was found dead on PPD3 (Day 42). (The single live born pup also died.)
• : Animal was found dead on GD22 (Day 39). Pregnancy was confirmed at necropsy.
• : Animal was found dead on GD21 (Day 38). Pregnancy was confirmed at necropsy.
• 4512: Animal was found dead on GD22 (Day 40). Pregnancy was confirmed at necropsy.

The hepatic effects were considered as cause of death of these animals.

There was no mortality in the Control, Low and Mid dose groups during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the surviving males, lower bodyweight gain was recorded during the first week of dosing in the High dose group. All other body weight or body weight gain values were normal in the surviving males in all dose groups. The body weights of the found dead or preterminally euthanized animals dramatically decreased before death.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption values correlated with the body weight changes.

In the surviving High dose males, lower food consumption was recorded in the beginning of the study, but after Day 14 it became normal. All other food consumption values were normal in the surviving males in the other dose groups. The food consumption of the found dead or preterminally euthanized High dose animals were significantly lower before death.

The control and test item-treated female animals had normal food consumption values during the study, except during the lactation period, where the High dose animals had slightly lower values (reaching statistical significance during PPD 4-13). The occasional statistically significantly higher values in the Mid dose group were considered incidental, not test item-related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly higher Mean Cell Volume (p<0.01) and Red Cell Distribution Width (p<0.05) were recorded in the female High dose group. No such differences were seen in the males. These differences were considered to be incidental, there was no clear relationship with dose and all recorded values were within the historical control ranges, also the key data of RBC and Haemoglobin were unaffected. These statistical differences were considered to not reflect an effect of the test item. The lymphocyte populations were all in the normal range with no indication of any treatment effect in any dose group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly higher aspartate aminotransferase activity (AST/GOT) (p<0.01) and alanine aminotransferase activity (ALT/GPT) (p<0.05) were measured in the male High dose group, reflecting hepatic damage seen at histology. Several other parameters linked to liver or kidney function also changed in individual male High dose animals, but without attaining statistical significance in the High dose group mean values, most probably because of the high individual variabilities in the data (total bilirubin, urea, ALKP, GGT, etc.). The High dose females had normal values. No test item-related findings were seen in the Low or Mid dose groups.

Statistical significances recorded in the bile acid and potassium concentrations were considered to be incidental, there was no clear relationship with dose and all recorded values were within the historical control ranges. These differences were considered not to reflect an effect of the test item.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared to the control, there were no test item-related statistically significant values recorded in any of the dose groups. Significantly lower urine pH (p<0.05) and higher urine protein level (p<0.05) were recorded in the female Mid dose group only. No such differences were seen in the males. These differences were considered to be incidental, there was no relationship with dose and all recorded values were within the normal biological ranges. These differences were considered to not reflect an effect of the test item.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

During the treatment (pre-mating and mating periods), the Control, Low and Mid dose animals had generally regular oestrus cycles, with normal incidence of pseudo-pregnancy or prolonged diestrus (1 to 3 out of the 12 animals).

In the High dose, 6/12 animals had irregular oestrus cycles. From these, 3/6 were pseudo-pregnancy (diestrus ≥10 days) and 2/6 were prolonged diestrus. In one case a rat had irregular cycles but without pseudo-pregnancy or prolonged diestrus. Six animals had regular cycles. This slightly increased incidence of irregular cycles might be test item-related, but possibly a secondary effect, related to the systemic toxicity of treatment. From the six High dose females with irregular cycles, two was found dead later (with confirmed implantations), two did not become pregnant despite positive mating, and two delivered normal litters.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The mating indices were 100% in all groups, except the High dose group where three males died before a successful mating could happen. The female fertility indices were 100% in the Control, Low and Mid dose groups and 75% in the High dose group (3 non-pregnant females out of the 12 animals). The gestation index was 100% in the Control, Low and Mid dose groups, meaning that all impregnated females in these groups delivered living pups in the study. The gestation index was 67% in the High dose, because from the 9 pregnant females 3 died during the gestation, surviving females had reproductive outcomes similar to historic controls. It was considered that there was no test item effect on reproductive parameters, although there was an effect on survival of the adults.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Remarks on result:

not measured/tested

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

In summary, daily administration of MDAC by oral gavage to Wistar rats at dose levels of 25, 100 or 400 mg/kg bw/day during the treatment period under the conditions of this study, caused mortality of 1 male and 4 females, and pre-terminal euthanasia of 3 males. Test item-related clinical signs were seen in the High dose group with very high individual differences. No adverse effects on body weight gain or food consumption were seen in any surviving animals. There were no changes recorded during the neurological assessment of any of the dose groups. Increased level of certain liver enzymes was seen in the High dose males, there were no other effects on the clinical pathology parameters.
The T4 hormone level was slightly decreased in the male High dose group, without any changes on thyroid weight or histopathology. Based on the very significant hepatotoxicity, the slight thyroid hormone difference was considered to be a secondary effect.
At necropsy, increased liver and kidney organ weights were recorded in the maleand female High dose groups, the liver of the Mid dose males and the kidney of the
Mid dose females were also slightly increased. The livers were discoloured and/orenlarged in the High dose group. At histopathology, massive hepatocellular
necrosis, multifocal bile duct hyperplasia and periportal fibrosis were seen in the High dose livers. No similar changes were seen in Mid dose livers of either sex.
Other histological changes seen only in early death animals were considered to be secondary to liver necrosis. There were no adverse test item-related macroscopic
or microscopic findings in the Low and Mid dose groups. Minor statistical differences in organ weights in Mid dose groups were considered to be adaptive
changes and not to reflect any adverse effects.
Because of the severe toxicity and unscheduled deaths in the High dose group, several reproductive parameters were altered, but all of these changes are
considered secondary and not a direct effect of the test item on the reproduction.
In the Low dose and Mid dose groups all parameters were normal, in surviving High dose animals, reproductive parameters were generally the same as controls.
Significantly more stillborn pups were recorded for the High dose group, mainly for dams that did not survive. The live born pups of the High dose group were born with significantly slightly lower bodyweights and their weight gains to day 13 was also lower. Besides this, there were no effects on the F1 offspring viability, clinical
signs, development or at observations following euthanasia. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital
distance, nipple retention, general development, thyroid gland weights, thyroid hormone level, etc.). The pups in the Low and Mid dose groups were completely
normal.
The NOAEL for Systemic toxicity for the adults was considered to be 100 mg/kg bw/day.
The NOAEL for Reproductive effects was considered to be 100 mg/kg bw/day.
The NOAEL for Pup development and survival was considered to be 100 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with theReproduction/Developmental Toxicity Screening Test in the Rats was to obtaininformation on the toxicity of the test item MDAC following repeated daily administration by oral gavage to Wistar rats. The study included a reproductive/developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day PPD13.

Parameters measured during the study included signs of morbidity and mortality twicedaily, daily general observation or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation, including

haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment, such as functional observation battery (FOB) including measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical

signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed.

For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups. Additionally, the liver and the lymphoid tissues (BALT, mandibular and mesenteric lymph nodes, spleen, thymus,

Peyer’s patch) of all animals were also microscopically examined.

In summary, daily administration of MDAC by oral gavage to Wistar rats at dose levels of 25, 100 or 400 mg/kg bw/day during the treatment period under the conditions of this study, caused mortality of 1 male and 4 females, and pre-terminal euthanasia of 3 males. Test item-related clinical signs were seen in the High dose group with very high individual differences. No adverse effects on body weight gain or food consumption were seen in any surviving animals. There were no changes recorded during the neurological assessment of any of the dose groups. Increased level of certain liver enzymes was seen in the High dose males, there were no other effects on the clinical pathology parameters.

The T4 hormone level was slightly decreased in the male High dose group, without any changes on thyroid weight or histopathology. Based on the very significant hepatotoxicity, the slight thyroid hormone difference was considered to be a secondary effect.

At necropsy, increased liver and kidney organ weights were recorded in the maleand female High dose groups, the liver of the Mid dose males and the kidney of the Mid dose females were also slightly increased. The livers were discoloured and/orenlarged in the High dose group. At histopathology, massive hepatocellular necrosis, multifocal bile duct hyperplasia and periportal fibrosis were seen in the High dose livers. No similar changes were seen in Mid dose livers of either sex. Other histological changes seen only in early death animals were considered to be secondary to liver necrosis. There were no adverse test item-related macroscopic or microscopic findings in the Low and Mid dose groups. Minor statistical differences in organ weights in Mid dose groups were considered to be adaptive

changes and not to reflect any adverse effects. Because of the severe toxicity and unscheduled deaths in the High dose group, several reproductive parameters were altered, but all of these changes are considered secondary and not a direct effect of the test item on the reproduction. In the Low dose and Mid dose groups all parameters were normal, in surviving High dose animals, reproductive parameters were generally the same as controls. Significantly more stillborn pups were recorded for the High dose group, mainly for dams that did not survive. The live born pups of the High dose group were born with significantly slightly lower bodyweights and their weight gains to day 13 was also lower. Besides this, there were no effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital

distance, nipple retention, general development, thyroid gland weights, thyroid hormone level, etc.). The pups in the Low and Mid dose groups were completely normal.

The NOAEL for Systemic toxicity for the adults was considered to be 100 mg/kg bw/day.

The NOAEL for Reproductive effects was considered to be 100 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 100 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

Significantly more stillborn pups were recorded for the High dose group, mainly for dams that did not survive. The live born pups of the High dose group were born with significantly slightly lower bodyweights and their weight gains to day 13 was also lower. Besides this, there were no effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, nipple retention, general development, thyroid gland weights, thyroid hormone level, etc.). The pups in the Low and Mid dose groups were completely normal.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

7

Species:

rat

Quality of whole database:

Klimisch 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

This substance will be not be classified as toxic to reproduction.

Significant systemic effects were seen at a dose level of 400 mg/kg/day and the reproductive and developmental effects were considered to be secondary to the systemic effects.  There were some systemic toxic effects observed at 100 mg/kg bw/day but the changes described are not considered to be significant (slight changes in liver and kidney weights) based on this

The NOAEL for Systemic toxicity for the adults was considered to be 100 mg/kg bw/day.

The NOAEL for Reproductive effects was considered to be 100 mg/kg bw /day.

The NOAEL for Pup development and survival was considered to be 100 mg/kg bw/day.

Reference to table 3.9.3 (EC 1272/2008) to assist in Category 2 classification gives a guidance value of 10<C≤100 mg/kg bw/day as a dose level range for classfication The classifications of Category 2 Repro 2 or  STOT Repeated Exposure Category 2 are not justified as no adverse (significant) effects were observed at either the 25 or 100 mg/kg bw/day dose levels.