Allyl phenoxyacetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (OECD 422), rat: NOAEL (fertility) ≥ 150 mg/kg bw/day in males and females

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Feb - 08 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks (males); 12 weeks (females)
- Weight at study initiation: 355.0 - 439.4 g (males); 242.3 - 295.3 g (females)
- Housing: During acclimation, pre-treatment, treatment, postmating and recovery period: 2 animals per cage; Mating period: 1 male and 1 female; Gestation period: 1 mated female; Lactation period: neonates were kept with the dam; animals were kept in stainless-steel cages (255W×465L×200H mm) and dams with pups in polycarbonate cages (260W x 420D x 180H mm) on aspen animal bedding
- Diet: Lab Diet® #5053 (PMI Nutrition International, USA) irradiated by gamma-ray, ad libitum
- Water: filtered, ultraviolet light-irradiated municipal tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: Microbial monitoring for diet was performed and a certificate of analysis for the diet was provided by the supplier. The drinking water was analyzed every 6 months for specified contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.8
- Humidity (%): 40.9 - 53.8
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil. Dose formulations were prepared at least one time per week and stored at room temperature. Dose formulations were transferred to the animal room at room temperature.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The verification of the dose level concentration was determined by gaschromatography prior to dosing. Samples were taken from the top, middle and bottom of all dose formulations. Results of dose formulations were 106.84, 102.04, and 101.53% at the dose levels of 7.5, 25, and 75 mg/mL respectively. They were acceptable as the mean concentration was within ±15% of the nominal concentration.

Duration of treatment / exposure:

Main groups:
males: at least 50 days, starting 2 weeks before mating, during mating until day prior to sacrifice
females: for 2 weeks prior to mating and continued until lactation day 13

Recovery groups:
at least 50 days; Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.

Frequency of treatment:

once daily, 7 days/week

Details on study schedule:

not applicable

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 (main groups)
6 (recovery groups; for control and high dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose level was selected based on the results of a repeated 2-week dose range-finding study in Sprague-Dawley rats in which doses of 15, 60 and 240/150 mg/kg bw/day have been tested. In that study, one found dead female was observed on treatment day 5 at dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased AST, ALT and yellow discoloration of liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, an increased AST and ALT were observed in males. Absolute and relative spleen weights were increased in both sexes. Based on the result of this dose range-finding study, 150 mg/kg bw/day was selected as the high dose, and 50 and 15 mg/kg bw/day were selected as the middle and the low dose, respectively. Animals of vehicle control were administered with the vehicle alone (corn oil).

- Rationale for animal assignment: Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and of group assignment), estrus cycle and for being free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality and morbidity observations were conducted twice daily except for the acclimation and recovery period where animals were observed once daily. In addition, it was conducted once on necropsy day.
- Cage side observations included: mortality/viability, clinical signs and general condition; during the gestation period: signs of abortion or premature delivery

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition

BODY WEIGHT: Yes
- Time schedule for examinations: first dosing day and then once per week; additionally on gestation days 0, 7, 14 and on lactation days 0, 4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption per animal [g food/animal/day] was calculated by subtracting the amount of residual feed from the amount presented

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER:
For haematology, clinical chemistry, neurobehavioural examination refer to IUCLID section 7.5.1

Oestrous cyclicity (parental animals):

A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating. Furthermore, regularity and length of the estrus cycle was examined during the treatment period until mating.
In addition, vaginal smear of sacrificed females were taken at termination to examine the stage of the estrus cycle and to allow a correlation with the histopathology of the female reproductive organs.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: thyroid hormone (T4) and the thyroid stimulating hormone (TSH) in blood on postnatal days 4 and 13

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on treatment day 50
- Maternal/female animals: on lactation day 14; non-parturition females after 27 days from final mating day
- Recovery group: at least 14 days after first scheduled sacrifice of main group males and females

GROSS PATHOLOGY: Yes
The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. Special attention was paid to the organs of the reproductive system. For lactating dams, the number of implantation sites (right and left) was counted. For non-parturition females, uterus implantation sites were examined after staining with ammonium sulfide according to KIT SOPs. Pregnancy was confirmed by implantation sites in the uterus.

ORGAN WEIGHTS: Yes
Paired organs were weighed together unless gross abnormalities were present. However, paired reproductive organs were weighed separately. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ/body weight ratios were calculated.
(for details see 7.5.1)

HISTOPATHOLOGY: Yes
Tissues shown in table 2 from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative, and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximately 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.
All tissues except for the reproductive organs collected from the first six animals per sex in the main group were further processed to slides, stained with hematoxylin and eosin (H&E), and examined microscopically. All reproductive organs collected from the main groups were further processed to slides, stained with H&E, and examined microscopically. Target organs from the recovery group animals were additionally evaluated microscopically.
(for details see 7.5.1)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at postnatal day 13.
- All animals were subjected to external postmortem examinations

GROSS NECROPSY
- Gross necropsy consisted of external examinations

Statistics:

Mean values and standard deviations were calculated. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using the Pristima System (Version 7.X. Xybion Medical Systems Corporation, USA) or Statistical Analysis Systems (SAS/STAT Version 9.2 and 9.4, USA). Data were considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data were analyzed using the Analysis of Variance (ANOVA), and the significance of inter-group differences were analyzed using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test, and the significance of inter-group differences was assessed between the VC and treated groups using the Dunn’s Rank Sum Test.
For comparing the control group with the recovery group, the data were analyzed for homogeneity for variance using the F-test. Homogeneous data were analyzed using the T-test and the significant difference was assessed between control and recovery group using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test and significant difference was assessed between control and recovery group using the Dunn’s Rank Sum Test.
One-way analysis of covariance (ANCOVA) was used to analyze pup body weights. The litter size was used as the covariate. Litter data were statistically evaluated using the statistical unit as a litter.
Data presented as frequencies were analyzed by χ2-test followed by the Fisher's exact test where necessary.

Reproductive indices:

- Precoital Time = No. of days taken to mate
- Mating index (%) = (No. of animals with evidence of mating / No. of animals paired) × 100
- Fertility index (%) = (No. of animals with impregnation or pregnancy / No. of animals paired) × 100
- Fecundity/Pregnancy Index (%) = (No. of animals with impregnation or pregnancy / No. of animals with evidence of mating) x 100
- Unaccounted-for sites (%) = (No. of implantation sites/litter) – (No. of pups born/litter) / No. of implantation sites/litter x 100
- Delivery Index = No. of dams with live pups on Day 0 of lactation / No. of pregnant dams x 100

Offspring viability indices:

- Viability Index = No. of live pups on Day 4 of lactation / No. of live pups at birth x 100
- Weaning Index = No. of live pups on Day 13 of lactation / No. of pups on Day 4 of lactation after culling x 100
- Sex ratio = (No. of male pups on PND 0/litter) / (No. of total pups on PND 0/litter) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related moribund animals were observed in any group throughout the study.
In males at 15, 50 and 150 mg/kg bw/day, salivation was observed in 11, 12, and 18 animals, respectively. In females at 15, 50, and 150 mg/kg bw/day, salivation was observed in 6, 12, and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have any dose-response.

In one female of control group found dead on gestation day 21 one early resorption, one late resorption and 7 dead fetuses were observed in the uterine examination. In the result of microscopic findings, moderate hemorrhage, slight alveolar edema and moderate perivascular congestion were observed in the lung. Moderate centrilobular necrosis in liver, right atrium dilatation in heart, slight atrophy in spleen and minimal mononuclear cell infiltration and hemorrhage in uterus were also observed. These were considered to be incidental since it was observed in the control group and there were no correlated microscopic changes in other animals in the same control group.

Non-parturition was observed in two females of control group and in one female at 150 mg/kg bw/day. Three females were subjected to unscheduled sacrifice on gestation day 27 and all of them were non-pregnant. In the results of microscopic findings, minimal squamous cell hyperplasia in stomach was observed in one animal at 150 mg/kg bw/day, but there were no other changes observed in the reproductive system in this animal.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related deaths occurred in any group throughout the study.
One female of control group was found dead on gestation day (GD) 21. Two females of control group and one female at 150 mg/kg bw/day were subjected to unscheduled sacrifice on gestation day 27 since they were non-pregnant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related changes in body weight and in body weight gain were observed in both sexes during the study. Statistically significant changes in body weight gain during the study were not considered test item-related since they were transient.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related changes in food consumption were observed in both sexes during the study.
A statistically significant change in food consumption during the study was not considered test item-related since there was no correlation with body weight changes and there was no dose-dependency.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males, total leukocyte count (WBC, 1.40-fold over control), absolute lymphocyte count (LYMA, 1.47-fold over control), absolute monocyte count (MONA, 1.43-fold over control), absolute large unstained cells counts (LUCA, 1.33-fold over control), fibrinogen (FIB, 1.22-fold over control) were increased at 150 mg/kg bw/day and platelet count (PLT, up to 1.21-folds over control) was increased at 50 and 150 mg/kg bw/day. The increases of WBC, LYMA, FIB and PLT were statistically significant. In females, WBC (1.30-fold over control), LYMA (1.37-fold over control), MONA (1.62-fold over control) and LUCA (1.58-fold over control) were increased at 150 mg/kg bw/day. The increases of WBC, MONA and LUCA were statistically significant. These changes recovered or showed a tendency of recovery at the end of the recovery period. Increased leukocyte counts, fibrinogen and platelet count in males and/or females at 150 mg/kg bw/day were correlated with inflammatory changes in the liver.
Other statistically significant changes were not considered test item-related, because these changes were minimal, there were no dose-relationships, and no correlations with microscopic changes and/or observed only in the recovery group.
(for result tables refer to 7.5.1)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, alanine aminotransferase (ALT, 2.80-fold over control), aspartate aminotransferase (AST, 1.46-fold over control), gamma glutamyl transpeptidase (GGT, 8.47-fold over control) and total bilirubin (TBIL, 1.07-fold over control) were increased at 150 mg/kg bw/day. The increases of ALT, GGT and TBIL were statistically significant. In females, ALT (2.05-fold over control), AST (1.43-fold over control), TBIL (1.21-fold over control) and GGT (8.48-fold over control) were also increased at 150 mg/kg bw/day. The increase of GGT was statistically significant. These changes recovered or showed a tendency to recover at the end of the recovery period. Increases of alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase and total bilirubin in males and/or females at 150 mg/kg bw/day were considered to be results of a massive damage of hepatocytes.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in functional behavior examinations were observed in both sexes during the study.
A statistically significant decrease in grip strength of forelimb in males at 50 and 150 mg/kg bw/day was not considered test item-related since there was no dose-dependency and no change in other parameters during the functional behavioral examinations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Test item-related changes were observed in liver, spleen, hepatic lymph node, and stomach of both sexes at 150 mg/kg bw/day.

Liver:
Slight to marked cholangiofibrosis, minimal to marked hydropic degeneration, minimal to marked necrosis, minimal to marked bile duct hyperplasia, minimal to slight hepatocyte hypertrophy and/or minimal to slight pigmented macrophages in both sexes and slight fibrosis in capsule in one male were observed at 150 mg/kg bw/day. These changes were observed in the periportal region except a fibrosis in capsule. After recovery period, cholangiofibrosis, hydropic degeneration and necrosis were fully recovered and bile duct hyperplasia and hepatocyte hypertrophy showed a tendency of recovery in both sexes at 150 mg/kg bw/day. In the 150 mg/kg bw/day recovery group, minimal to moderate periportal fibrosis in both sexes were also observed and pigmented macrophage was similar to the main groups in both sexes.
Cholangiofibrosis, hydropic degeneration, and necrosis were considered to be an adverse effect of the test-item and are well known and often observed as hepatocellular toxicity caused by various xenobiotics and chemicals. Periportal fibrosis was observed and shown to be a recovery reaction.
Bile duct hyperplasia, hepatocyte hypertrophy, pigmented macrophages and/or fibrosis in the capsule accompanied the above named findings were not considered as an adverse effect at the severity as seen here. These changes were considered to be adaptive changes to a drug-induced increase in a metabolic workload or were considered as secondary findings related to the aforementioned adverse effects in the liver.
Spleen and hepatic lymph node:
Minimal to slight lymphoid hyperplasia in both sexes at 150 mg/kg bw/day and slight chronic inflammation in the capsule in one female at 150 mg/kg bw/day were observed. At the end of the recovery period and lymphoid hyperplasia showed a tendency of recovery in both sexes at 150 mg/kg bw/day and a capsular fibrosis in both sexes and chronic inflammation in capsule were observed in females at 150 mg/kg bw/day. In the hepatic lymph node, moderate lymphoid hyperplasia, slight congestion were observed in both sexes at 150 mg/kg bw/day.
These changes in spleen and hepatic lymph nodes were considered to be associated with a secondary response to the inflammatory changes in the liver.

Stomach:
Minimal to slight squamous cell hyperplasia in the non-glandular region was observed in both sexes at 150 mg/kg bw/day and fully recovered after the recovery period. This change was considered to be non-adverse but an adaptive response following a local irritation caused by the test item since it fully subsided after the recovery period.

Other changes observed in microscopic findings were considered as incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among the control and the treated groups.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in the adult male animals.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

A decreased absolute weight of testes and epididymides in males at 150 mg/kg bw/day did not represent a meaningful toxicologic finding as there were no correlated microscopic findings and/or it fully recovered during the recovery period.

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to the highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

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):

A statistically significant decrease in body weight of the F1 pups was observed on PND 13 (male pups: 92% of VC, female pups: 91% of VC) at 150 mg/kg bw/day.
(see table 1)

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:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in pups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at 50 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1. Pups body weight (males)

Group		Control	15 mg/kg bw/day	50 mg/kg bw/day	150 mg/kg bw/day
PND 0	Mean	7.04	6.75	6.96	7.05
	SD	0.44	0.65	0.35	0.50
	Covariate adjusted mean	6.97	6.79	7.01	7.01
	n	9	12	12	11
PND 4 (preculling)	Mean	11.52	11.11	11.08	10.71
	SD	1.25	1.69	0.97	0.85
	Covariate adjusted mean	11.35	11.08	11.28	10.65
	n	9	12	12	11
PND 4 (postculling)	Mean	11.82	11.38	11.51	11.02
	SD	1.32	1.63	0.99	0.83
	n	9	12	12	11
PND 13	Mean	35.78	35.13	35.05	32.32+D
	SD	1.46	2.93	1.83	2.68
	Covariate adjusted mean	35.51	34.98	35.24	32.51*D
	n	9	12	12	11

+D = Dunnett LSD Test Significant at the 0.01 level or at 0.05 level (*)   

Table 2. Pups body weight (females)

Group		Control	15 mg/kg bw/day	50 mg/kg bw/day	150 mg/kg bw/day
PND 0	Mean	6.58	6.32	6.646.96	6.637.05
	SD	0.41	0.63	0.38	0.53
	Covariate adjusted mean	6.51	6.36	6.69	6.59
	n	9	12	12	11
PND 4 (preculling)	Mean	10.82	10.46	10.66	10.26
	SD	1.27	1.67	0.97	0.98
	Covariate adjusted mean	10.64	10.44	10.88	10.20
	n	9	12	12	11
PND 4 (postculling)	Mean	10.99	10.72	11.02	10.48
	SD	1.19	1.67	1.11	1.10
	n	9	12	12	11
PND 13	Mean	34.09	33.60	34.19	30.64*R
	SD	1.46	3.27	1.71	3.64
	Covariate adjusted mean	33.86	33.47	34.36	30.80*D
	n	9	12	12	11

* R = Dunn Rank Sum Test Significant at the 0.05 level+D = Dunnett LSD Test Significant at the 0.01 level or at 0.05 level (*)  

Conclusions:

The NOAEL for parental systemic toxicity was considered to be 50 mg/kg bw/day in both sexes, based on cholangiofibrosis, hydropic degeneration and necrosis of liver in both sexes at 150 mg/kg bw/day. The NOAEL for parental fertility was considered to be 150 mg/kg bw/day, since no adverse effects were observed on parental reproductive function.
The NOAEL for developmental toxicity was considered to be 50 mg/kg bw/day based on decreased pup body weights on postnatal day 13 at 150 mg/kg bw/day. This effect was considered to be a secondary effect of parental systemic toxicity. No further effect on development was observed.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2018). Twelve Sprague Dawley rats per sex and dose were treated via gavage with test substance at doses of 15, 50 and 150 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, non-mated recovery groups of 6 rats per sex were allocated to the control and high dose groups. Males and females of the main groups were dosed for two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day (LD) 13 in females. The high-dose recovery group was assigned to a two week treatment free period. The dose levels were selected based on the results of a repeated dose 2-week dose range-finding study in which doses of 15, 60 and 240/150 mg/kg bw/day had been tested. In that study, one found dead female was observed on treatment day 5 at dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased alanine aminotransferase (AST), alanine aminotransferase (ALT) and yellow discoloration of the liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, increased AST and ALT levels were observed in males. Absolute and relative spleen weights were increased in both sexes.

In the main study, the following parameters for reproductive function were analyzed: estrus cycle, precoital time, fertility data (mating, fertility, fecundity and pregnancy index) and reproductive littering findings. In gross pathology of parental animals special attention was paid to reproductive organs.

Hepatocellular toxicity was observed in parental animals consisting of cholangiofibrosis, hydropic degeneration and necrosis of the liver with associated effects of increases of alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase and total bilirubin in blood and secondary responses of lymphoid hyperplasia in spleen and hepatic lymph nodes at 150 mg/kg bw/day.

None of the parameters on reproduction showed any treatment related sign of toxicity in any dose group and both sexes.

In males, a decreased absolute weight of testes and epididymides was observed at 150 mg/kg bw/day. This change was considered not toxicologically relevant since no correlated microscopic findings were observed and the organ weights recovered during the recovery period.

In one female of the control group found dead on gestation day 21 one early resorption, one late resorption and 7 dead fetuses were observed in the uterine examination. At microscopic examination, moderate hemorrhage, slight alveolar edema and moderate perivascular congestion were observed in the lung. Moderate centrilobular necrosis in liver, right atrium dilatation in heart, slight atrophy in spleen and minimal mononuclear cell infiltration and hemorrhage in uterus were also observed. These findings were considered to be incidental since they were observed in the control group and there were no correlated microscopic changes in other animals of the same control group.

Non-parturition was observed in two females of the control group and in one female at 150 mg/kg bw/day. Three females were subjected to unscheduled sacrifice on gestation day 27 since all of them were non-pregnant. At microscopic examination, minimal squamous cell hyperplasia in stomach was observed in one animal at 150 mg/kg bw/day, but there were no changes observed in the reproductive system in this animal.

In conclusion, the NOAEL for fertility of parental animals was considered to be ≥ 150 mg/kg bw/day, since no adverse effects on fertility parameters were observed up to the highest dose tested. The NOAEL for systemic toxicity in parental animals was considered to be 50 mg/kg bw/day, based on cholangiofibrosis, hydropic degeneration and necrosis of the liver in both sexes.

Effects on developmental toxicity

Description of key information

Oral (OECD 422), rat: NOAEL (development) 50 mg/kg bw/day in males and females

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

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

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2018). Twelve Sprague Dawley rats per sex and dose were treated via gavage with test substance at doses of 15, 50 and 150 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, non-mated recovery groups of 6 rats per sex were allocated to the control and high dose groups. Males and females of the main groups were dosed for two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day (LD) 13 in females. The high-dose recovery group was assigned to a two week treatment free period. The dose levels were selected based on the results of a repeated dose 2-week dose range-finding study in which doses of 15, 60 and 240/150 mg/kg bw/day had been tested. In that study, one found dead female was observed on treatment day 5 at a dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased AST, ALT and yellow discoloration of the liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, increased AST and ALT levels were observed in males. Absolute and relative spleen weights were increased in both sexes.

In the main study, pups were observed for mortality, clinical signs, body weight, sexual maturation (anogenital distance and nipple retention), thyroid hormones (T4 and TSH) in blood and macroscopic abnormalities after scheduled sacrifice on postnatal day 13.

A statistically significant decrease in body weight of the F1 pups was observed on PND 13 (male pups: 92%, female pups: 91%) at 150 mg/kg bw/day. No other test substance related findings on development were observed in pups in any dose group.

In conclusion, decreased body weights of pups on postnatal day 13 at 150 mg/kg bw/day were considered to be adverse and a secondary effect to the systemic liver toxicity in the dams. Therefore, the NOAEL for developmental toxicity was considered to be 50 mg/kg bw/day.

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification. Reduced pup body weights are considered to be a secondary effect of parental systemic toxicity and thus were not considered for classification on developmental toxicity.