4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined with a repeated dose toxicity and an assessment of genotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 09, 2015 to December 02, 2015

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:

but considered to have not affected the integrity of the study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- A total of 130 Hsd: Sprague Dawley SD rats (65 males and 65 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, were ordered from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy. 5 animals per cage.
- Acclimatisation period of approximately 3 to 9 weeks, depending on the type of treatment.
- Temperature and relative humidity: 22+/- 2°C and 55 +/- 15%, respectively.
- Artificial light for 12 hours.
- Feed (commercially available laboratory rodent diet (4 RF 21) and water: ad libitum.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test item was administered orall by gavage to animals at 5 mL/kg bw.

Details on mating procedure:

During mating, animals were housed one male to one female in clear polycarbonate cages with a stainless steel mesh lid and floor, as indicated in the relevant SOP. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating, the females were transferred to individual solid bottomed cages, as indicated in the relevant SOP, for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed to confirm that the proposed formulation procedure, in the range of 10-120 mg/mL, was acceptable (concentration and homogenicity) and that the stability of the formulation was satisfactory (RTC Study No. A0769).

Duration of treatment / exposure:

Males:
- 0 and 100 mg/kg bw/day groups: animals were dosed once a day, 7 days a week,for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter until the minimum total dosing period of 28 days had been completed including the day before necropsy. They were treated for a total of 29 days.
- 350-50 mg/kg bw/day group: animals were treated for a total of 38 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (for at least 40 days for 0 and 100 mg/kg bw/day groups and 42 days for the 350-50 mg/kg bw/day group) or the day before sacrifice (for not pregnant female). Dose volumes were adjusted once per week for each animal according to the last recorded bodyweight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day1 post partum. Thereafter individual dose volumes remained constant.
- The treatment of the 600 mg/kg bw/day group was stopped after 6 days of treatment and animals were sacrificed.
- The treatment of the 250 mg/kg bw/day was stopped after 21 days of treatment and animals were sacrificed.
Recovery groups:
Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks. No treatment was given during the recovery period of 2 weeks.

Frequency of treatment:

Once a day

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

This level was stopped after 3 weeks of treatment and animals were sacrificed

Dose / conc.:

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

Remarks:

This level was stopped after 6 days of treatment and animals were sacrificed

Dose / conc.:

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

Remarks:

This level was stopped after 3 days of treatment and animals were allowed a 4-day wash-out period

Dose / conc.:

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

Remarks:

This level was given after a 4-day wash-out period in the group 350 mg/kg bw/day

No. of animals per sex per dose:

10 for treated groups with the test substance
5 for recovery and positive control groups

Control animals:

yes, concurrent vehicle

Details on study design:

The original dose levels of 100, 250, 350-50, and 600 mg/kg bw/day were selected by the Sponsor based on information from previous studies.

Parental animals: Observations and examinations:

- Mortality: every day (early or mid day).
- Clinical signs: at the same time interval each day (observation of the cage tray was performed starting from the first detectable sign). The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic ortonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size,unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
- Grip strength and sensory reactivity to stimuli; motor activity assessment (main and recovery groups): once during the study.
- Body weight and food consumption: weekly (+ dams were also weighed on Days 1 and 4 post partum).
- Clinical pathology investigations (samples of blood: haematology, coagulation test, chemistry)

Sperm parameters (parental animals):

A qualitative examination of the testes was performed in 5 randomly selected control and 100 mg/kg bw/day dose group males (stages of the spermatogenic cycle, missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen).

Litter observations:

- Pups identification, weight and observation: once daily

Postmortem examinations (parental animals):

- Necropsy, organ weights, tissue fixation and preservation, histopathological examinations

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. The criterion for statistical significance was p<0.05. The mean values standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

- Vaginal smears, mating (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray) and parturition and gestation length.

Offspring viability indices:

- Mammalian erythrocyte micronucleus test: samples of bone marrow were collected between 18 and 24 hours following the final treatment and approximately 48 hours following the second last treatment.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Signs of toxicity such as decreased activity, piloerection, kyphosis, staining of the perianal region/muzzle and pallor were seen in almost all animals receiving 250 mg/kg bw/day, during the first week of mating period and in almost all animals receiving 600 mg/kg bw/day, during the first week of treatment.
- Piloerection, kyphosis and/or decreased activity and staining of the perianal region/muzzle were seen in 2 females when receiving 350-50 mg/kg bw/day; no clinical signs were seen in males and females when receiving 50 mg/kg bw/day. Males receiving 100 mg/kg bw/day did not show any clinical signs throughout the study. Kyphosis, piloerection, dyspnoea and/or pallor, swollen ventral region of the neck and/or staining of the perianal region, decreased activity, cold to touch were the major signs of toxicity seen in 3 females which were sacrificed for humane reasons, on Day 1 post partum.
- Recovery groups: No clinical signs were seen in treated animals throughout the study, during treatment and recovery periods.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- Two males receiving 600 mg/kg bw/day were found dead on Day 5 and 7 of treatment. Kyphosis, piloerection and/or staining on the perianal region were seen the day before death. The remaining animals of this group were killed after 6 days of treatment.
- Five males and 6 females receiving 250 mg/kg bw/day showed kyphosis, piloerection, pallor and/or decreased activity, staining of perianal region, swollen neck and cold to touch and were killed for humane reasons. The remaining animals in this group were killed after 21 days of treatment.
- Four unscheduled deaths occurred in females receiving 100 mg/kg/day. One female was found dead and 3 females were killed for humane reasons on Day 1 post partum. The clinical signs observed in these last three females were similar to those observed in humanely killed animals of the 250 and 600 mg/kg bw/day groups, suspended due to high toxicity.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the study:
- No relevant differences in body weight and body weight gain were noted between control and treated groups, of both sexes, during the study, with the exception of statistically significant decreases in body weight and body weight gain, seen in the 100 mg/kg bw/day group of females when compared to controls, during the post partum period (-13% and -4 fold on Day 4, respectively).
- Recovery groups: during treatment, slight decrease in body weight gain was seen on Day 8 of treatment, in the 100 mg/kg bw/day group of males (statistically significant) and females, when compared to controls. During recovery, no relevant differences in body weight and body weight gain were noted between control and treated group, of both sexes.
Terminal body weight:
- Terminal body weight was unaffected by treatment in males, while a slight statistically significant decrease (13%) in terminal body weight was seen in females (100 mg/kg bw/day), when compared to controls. Terminal body weight was also unaffected by treatment in both sexes of recovery groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Slight statistically significant decrease in food consumption was recorded in females dosed at 100 mg/kg bw/day on Day 7 post coitum (-6%) and on Day 4 post partum (-47%), compared to controls. No changes were noted in treated males during treatment.
- Recovery groups: no relevant differences in food consumption were noted in recovery group animals (both sexes), during the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- When compared with controls, reticulocytosis was recorded in treated males (47%) with no relevant changes of erythrocytes/haemoglobin. In addition, treated females showed a slight decrease of haemoglobin and haematocrit (approximately 9%) and increase of platelets (38%). Due to the slight magnitude, these changes were not considered to be adverse. The differences of leucocytes between control and treated females were mainly due to the high values of one control animal.
- Recovery groups: changes recorded during the dosing phase were no longer observed, confirming complete reversibility.
- Coagulation: no changes were recorded.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Fluctuations of liver/metabolic markers were recorded in some treated animals. Compared with mean control data, 1 male showed increase of alanine aminotransferase (64%), aspartate aminotransferase (3.5 fold), bilirubin (6.3 fold) and bile acids (2.5 fold) and another one showed increase of cholesterol (78%) and triglycerides (93%). An increase of aspartate aminotransferase (2.1fold), urea (59%) and a decrease of triglycerides (74%) was observed in one female, and an increase of phosphorus (34%) was detected in another female.
- Recovery groups: full recovery was observed for all changes recorded during the dosing phase.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- During the last week of treatment for in the 250 mg/kg bw/day group (Week 3), females showed slow handling reactivity at the removal from the cages; in the open arena hunched gait and/or mobility impairment was seen in males and females.
- In the 0, 50, and 100 mg/kg bw/day groups: neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test substance, with the exception of one female of the 100 mg/kg bw/day group which showed slow handling reactivity at the removal from the cage and moderately hunched gait in the open arena.
- No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic observations:
- Final sacrifice
Groups 250 and 600 mg/kg bw/day (sacrificed before term): Few males and females showed minimal to moderate congestion in the gastro intestinal tract and lymphocytolysis of spleen and thymus in a single female.
Groups 100 and 350-50 mg/kg bw/day (sacrificed at term). Treatment-related changes in females, consisting in minimal to marked atrophy of thymus, were observed. No treatment-related histopathological changes were observed in males.
- Recovery sacrifice
No histopathological changes were noted in the thymus of control and treated females sacrificed after 2 weeks of recovery period.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle, reproductive parameters, pairing combination and mating performance:
- All males, with the exception of 2 males in the 350-50 mg/kg bw/day group, and all females mated. One control male did not induce pregnancy. Oestrous cycle and reproductive parameters did not show differences related to treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic cycle:
- In control and 100 mg/kg bw/day group males, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

Oestrous cycle, reproductive parameters, pairing combination and mating performance:
- All males, with the exception of 2 males in the 350-50 mg/kg bw/day group, and all females mated. One control male did not induce pregnancy. Oestrous cycle and reproductive parameters did not show differences related to treatment.

- Fate of females: One control female was found not pregnant at necropsy. The number of females with live pups on Day 4 post partum was 9 in the control, 10 in the low dose group and 6 in the high dose group.
- Implantation, pre-birth loss data and gestation length of females:
Gestation periods were similar between treated animals and controls being a mean of 22 days. No relevant differences were observed in number of corpora lutea and implantation, pre-implantation and pre-natal loss between control and treated females.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced number of females with live pups in the group 100 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Reproductive and developmental parameters

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Cold to touch, small, apparently no food intake (milk) were the main clinical signs noted in control and treated pups. In addition, found dead or missing pups were observed both in control and treated groups, with the same incidence.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Litter data at birth, on Day 1 and on Day 4 post partum of females:
- Statistically significant lower mean pup weight at Day 1 post partum, litter weight and mean pup weight at Day 4 post partum, were seen in females receiving 100 mg/kg bw/day.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sex ratio of pups:
- Sex ratios at birth and on Day 4 post partum did not show differences between treated and control groups.
Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum:
- Pups of humanely killed females dosed at 100 mg/kg bw/day were sacrificed on Day 1 post partum. No milk in the stomach of all pups from one female and scab on the muzzle of one pup from another female were seen at necropsy. The number of decedent pups was comparable between groups. No milk in stomach was recorded for few pups in the 100 mg/kg bw/day group as well as in the control group. For many pups, all organs autolysed in abdominal and/or thoracic cavities were noted at necropsy. Few pups were missing during the observation period in control and treated groups. No signs were seen in pups sacrificed on Day 4 post partum.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Developmental effects

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: bodyweight

Organ:

other: bodyweight

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

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:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

Based on the results, the reproductive and developmental NOAELs for the study were both considered to be 50 mg/kg/day (i.e. 47.4 mg a.i./kg bw/day).

Executive summary:

A study was conducted to determine the reproductive and developmental toxicity of the test substance (in the form of a dark brown powder of 94.8% purity) according to OECD Guideline 422. Male and female Sprague Dawley rats (10/sex/dose for the main study and 5/sex/dose for the recovery groups) were exposed to the following concentrations (dissolved in polyethylene glycol, at a constant volume of 10 mL/kg bw):

 

Group	Dose (mg/kg bw/day)	Treatment period	Comment	Recovery?
1	0	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 29 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day3 post-partum: 41-51 days	Controls	Yes
2	100	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 29 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day3 post-partum: 41-51 days	-	Yes
3	250	-	This level was stopped after 3 weeks of treatment and animals were sacrificed	No
4	600	-	This level was stopped after 6 days of treatment and animals were sacrificed	No
8	350	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 38 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day3 post-partum: 49-64 days	This level was stopped after 3 days of treatment and animals were allowed a 4-day wash-out period	No
8	50		This level was given after a 4-day wash-out period	No

 

Animals in the controls and 100 mg/kg bw/day groups were assigned to a 2 week recovery period after end of treatment.

 

In the main groups, mortality and clinical signs were recorded daily. Neurotoxicity, motor activity, grip strength and sensory reactivity to stimuli were assessed once during the study. Body weight and food consumption were measured weekly (and dams were also weighed on Days 1 and 4 post-partum).

 

Vaginal smears were taken daily in the morning starting two weeks before pairing throughout the mating period until a positive identification of copulation was made. The vaginal smear data were examined to determine anomalies of the oestrous cycle and the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation. A parturition check was performed from Day 20 to Day 25post-coitum. One female which did not give birth after 25 dayspost-coitumperiod was sacrificed on Day 28 post-coitumand was found not pregnant at necropsy. Gestation length was calculated as the time between the day of successful mating (Day 0post-coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage was considered Day 0post-partum. As soon as possible after parturition was considered complete, all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4post-partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.

 

At necropsy, blood samples were collected for haematology and clinical chemistry investigations. Also, a gross macroscopical examination was conducted, and organs were weighed. Routine histopathological examination was performed on control and high dose (100 mg/kg bw/day) animals (5/sex/group randomly selected) and on 250 mg/kg bw/day animals. In addition, thymus was examined in all females at 0, 100 and 350-50 mg/kg bw/day. Evaluation of body weight, clinical signs and macroscopic observations of pups was also performed.

 

Animals of the recovery groups were treated for a total of 4 consecutive weeks and sacrificed after 2 weeks of recovery. The following parameters were evaluated in these animals: clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction tostimuli), body weight, food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological evaluation of the thymus in all females, only.

 

Based on the results obtained on the parental generation, the systemic NOAEL for the study was considered to be 100 mg/kg/day (i.e. 94.8 mg a.i./kg bw/day).

 

All males, with the exception of 2 males in Group 8, and all females mated. One control male did not induce pregnancy. Oestrous cycle and reproductive parameters did not show differences related to treatment.

 

Gestation periods were similar between treated animals and controls being a mean of 22 days. No relevant differences were observed in number of corpora lutea and implantation, pre-implantation and pre-natal loss between control and treated females.

 

Statistically significant lower mean pup weight at Day 1post-partum, litter weight and mean pup weight at Day 4post-partum, were seen in females receiving 100 mg/kg/day. Sex ratios at birth and on Day 4post-partumdid not show differences between treated and control groups.

 

Cold to touch, small, apparently no food intake (milk) were the main clinical signs noted in control and treated pups. In addition, found dead or missing pups were observed both in control and treated groups, with the same incidence.

 

Pups of humanely killed 100 mg/kg bw/day females were sacrificed on Day 1post-partum: no milk in stomach in all pups of one female and scab on the muzzle of one pup of a single female were seen at necropsy. The number of decedent pups was comparable between groups. No milk in stomach was recorded for few pups at 100 mg/kg bw/day as well as in the control group. For many pups, all organs autolysed in abdominal and/or thoracic cavities were noted at necropsy. Few pups were missing during the observation period in control and treated groups. No signs were seen in pups sacrificed on Day 4post-partum.

 

Based on these results, the reproductive and developmental NOAELs for the study were both considered to be 50 mg/kg/day (i.e. 47.4 mg a.i./kg bw/day) (Rossiello, 2016).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

47.4 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the reproductive and developmental toxicity of the test substance (in the form of a dark brown powder of 94.8% purity) according to OECD Guideline 422. Male and female Sprague Dawley rats (10/sex/dose for the main study and 5/sex/dose for the recovery groups) were exposed to the following concentrations (dissolved in polyethylene glycol, at a constant volume of 10 mL/kg bw):

 

Group	Dose (mg/kg bw/day)	Treatment period	Comment	Recovery?
1	0	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 29 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day 3 post-partum: 41-51 days	Controls	Yes
2	100	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 29 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day3 post-partum: 41-51 days	-	Yes
3	250	-	This level was stopped after 3 weeks of treatment and animals were sacrificed	No
4	600	-	This level was stopped after 6 days of treatment and animals were sacrificed	No
8	350	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 38 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day 3 post-partum: 49-64 days	This level was stopped after 3 days of treatment and animals were allowed a 4-day wash-out period	No

 

Animals in the controls and 100 mg/kg bw/day groups were assigned to a 2 week recovery period after end of treatment.

 

In the main groups, mortality and clinical signs were recorded daily. Neurotoxicity, motor activity, grip strength and sensory reactivity to stimuli were assessed once during the study. Body weight and food consumption were measured weekly (and dams were also weighed on Days 1 and 4 post-partum).

 

Vaginal smears were taken daily in the morning starting two weeks before pairing throughout the mating period until a positive identification of copulation was made. The vaginal smear data were examined to determine anomalies of the oestrous cycle and the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation. A parturition check was performed from Day 20 to Day 25post-coitum. One female which did not give birth after 25 dayspost-coitumperiod was sacrificed on Day 28 post-coitumand was found not pregnant at necropsy. Gestation length was calculated as the time between the day of successful mating (Day 0post-coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage was considered Day 0post-partum. As soon as possible after parturition was considered complete, all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4post-partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.

 

At necropsy, blood samples were collected for haematology and clinical chemistry investigations. Also, a gross macroscopical examination was conducted, and organs were weighed. Routine histopathological examination was performed on control and high dose (100 mg/kg bw/day) animals (5/sex/group randomly selected) and on 250 mg/kg bw/day animals. In addition, thymus was examined in all females at 0, 100 and 350-50 mg/kg bw/day. Evaluation of body weight, clinical signs and macroscopic observations of pups was also performed.

 

Animals of the recovery groups were treated for a total of 4 consecutive weeks and sacrificed after 2 weeks of recovery. The following parameters were evaluated in these animals: clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction tostimuli), body weight, food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological evaluation of the thymus in all females, only.

 

Based on the results obtained on the parental generation, the systemic NOAEL for the study was considered to be 100 mg/kg/day (i.e. 94.8 mg a.i./kg bw/day).

 

All males, with the exception of 2 males in Group 8, and all females mated. One control male did not induce pregnancy. Oestrous cycle and reproductive parameters did not show differences related to treatment.

 

Gestation periods were similar between treated animals and controls being a mean of 22 days. No relevant differences were observed in number of corpora lutea and implantation, pre-implantation and pre-natal loss between control and treated females.

 

Statistically significant lower mean pup weight at Day 1 post-partum, litter weight and mean pup weight at Day 4 post-partum, were seen in females receiving 100 mg/kg/day. Sex ratios at birth and on Day 4 post-partum did not show differences between treated and control groups.

 

Cold to touch, small, apparently no food intake (milk) were the main clinical signs noted in control and treated pups. In addition found dead or missing pups were observed both in control and treated groups, with the same incidence.

 

Pups of humanely killed 100 mg/kg bw/day females were sacrificed on Day 1 post-partum: no milk in stomach in all pups of one female and scab on the muzzle of one pup of a single female were seen at necropsy. The number of deceased pups was comparable between groups. No milk in stomach was recorded for few pups at 100 mg/kg bw/day as well as in the control group. For many pups, all organs autolysed in abdominal and/or thoracic cavities were noted at necropsy. Few pups were missing during the observation period in control and treated groups. No signs were seen in pups sacrificed on Day 4 post-partum.

 

Based on these results, the reproductive and developmental NOAELs for the study were both considered to be 50 mg/kg/day (i.e. 47.4 mg a.i./kg bw/day) (Rossiello, 2016).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

47.4 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Based on the results of a combined repeated dose oral toxicity study with reproductive/developmental toxicity screening in rat, no classification for reproductive/developmental toxicity is required for the test substance according to CLP (EC 1272/2008) criteria.