Reaction mass of isopropyl dihydrogen phosphate and phosphoric acid and propan-2-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Thstructural analogue , Hordaphos CC MIS was evaluated for possible adverse effects following repeated oral dosing for relatively limited period of time and to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development. The study was performed according to OECD 422.

 

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to main and recovery groups. Each main group (G1, G2, G3and G4) consisted of 12 males and 12 females and recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R groups were administered with vehicle [Distilled water], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered at the dose volume of 10 mL/kg body weight.

The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 4 after which the pups were sacrificed on lactation Day 4 and females (dams) were sacrificed on lactation day 5 after overnight fasting (water allowed). The recovery group animals were treated till first scheduled sacrifice of dam(s) and observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

 

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis, organ weights (both absolute and relative) were observed in both the sex.

 

The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights.

 

The animals (main group and recovery) did not reveal any treatment related gross as well as histopathological changes on examination.

 

Conclusions

For reproductive and developmental toxicity assessment, the No Observed Adverse Effect Level (NOAEL) of the test item Hordaphos CC MIS was found to be 1000 mg/kg body weight, based on no adverse effects on the parameters like copulatory interval, gestation length, gestation/lactation body weight, gestation/lactation feed consumption, litter weights, mean pup weights, uteri observations and no gross pathological findings in both dams and pups.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2015 to 23 October 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:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: Inhouse bred animals
- Age at study initiation: (P) 9 to 10 wks
- Weight at study initiation: (P) Males: 189.99 209.29 g; Females: 180.09 198.53 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.

i. Pre mating
Two animals of same sex and group per cage were housed.

ii. Mating
During mating, two animals (one male and one female) of same group were housed.

iii. Post mating
After confirming presence of sperm in the vaginal smear and (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet Pellet (Manufactured by Harlan Laboratories)
- Water (e.g. ad libitum): Deep borewell water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter with purifier was provided in plastic water bottles with stainless steel sipper tubes
- Acclimation period: Healthy and young adult animals were acclimatized for five days to experimental room conditions before initiation of treatment (08 July 2015 to 12 July 2015)

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19.8 to 22.6
- Humidity (%): 49 to 64
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 08 July 2015; To: 11 September 2015

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Test item formulations were prepared daily before administration. The required quantity of test item was weighed into a clean glass beaker and there by adding little volume of vehicle into the beaker, mixed well with glass rod and transferred into the measuring cylinder. Again a small quantity of vehicle was added, mixed well with glass rod to rinse the beaker and transferred into the measuring cylinder. The rinsing procedure was repeated to
ensure complete transfer of the test item formulation into a measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE

- Justification for use and choice of vehicle (if other than water): The test item was clearly miscible in distilled water as evidenced by the inhouse miscibility test. Hence, distilled water was selected for the test item formulation preparation.
- Concentration in vehicle: 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): 10 mL/kg body weight.
- Lot/batch no. (if required): Distilled Water Batch No.: 5108, Expiry Date: 06/2016; Batch No.: 5089, Expiry Date: 07/2016; Batch No.: 5103, Expiry Date: 08/2016; Batch No.: 5193., Expiry Date: 08/2016.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Two weeks and the females with unsuccessful pairing within 14 days were remated with proven males of same group.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Sperm in vaginal smear.
- After 14 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 (how): Housed individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis for dose concentration verification of Hordaphos CC MIS was done by Analytical Chemistry of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 116 and the results were presented as appendix 28. Sampling and analysis of formulations were performed on first day of treatment and during week 5 (Day 33) of the treatment. The samples were collected in duplicates from each concentration at volume of 5 mL (5 mLx2).

The collected samples were transferred to Analytical Chemistry of Bioneeds India Private Limited for dose concentration analysis. One set of aliquots of each formulation were analyzed. The second aliquots were stored as a back up purpose at room temperature. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) was equal to or less than 10.0%.

Duration of treatment / exposure:

The test item or vehicle was administered to animals through oral (gavage) route once daily.

The main group males were treated for two weeks premating, during mating and up to the day before sacrifice during postmating period (total of 35 days of treatment).

The main group females were treated for two weeks premating period, during mating, pregnancy (gestation) and up to lactation day 4 after which the pups were sacrificed on lactation Day 4 and females (dams) were sacrificed on lactation day 5 after overnight fasting (water allowed).

The recovery group animals were treated till first scheduled sacrifice of dam(s) and were observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

The test item/vehicle was administered by oral (gavage) route using intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 10 mL/kg with the concentration of 10, 30 and 100 mg/mL for low, mid and high dose/high dose recovery groups, respectively. Distilled water was administered to vehicle control/vehicle control recovery group at an equivolume of 10 mL/kg body weight. The actualdose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 10 weeks

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Main Group: 24 (12 Males + 12 Females) per dose
Recovery Group: 10 (5 males + 5 females) per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/ high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item Hordaphos CC MIS when administered through oral (gavage) for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1022).

These doses were selected as the test item Hordaphos CC MIS when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (50, 300 and 800 mg/kg body weight).

Positive control:

Not Applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Time schedule: All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment and recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group and recovery group males and females were weighed on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and within 24 hours of parturition (day 1 postpartum) and on day 4 postpartum during lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

Oestrous cyclicity (parental animals):

Not Applicable

Sperm parameters (parental animals):

Not Applicable

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities for born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals [after completion of 35 days of treatment.]
- Maternal animals: All animals [on lactation day 5]

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 [Section 6.16.1 in Study Report] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
The offspring were sacrificed on their lactation day 4 (LD 4).
The sacrificed pups and dead pups were examined for gross abnormalities and the findings were recorded.

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

Statistics:

The data was subjected to various statistical analyses using SPSS software version 22.

All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests are designated by the superscripts throughout the report as stated below:

* Statistically significant (P<0.05) change than the vehicle control group.
Note: Data of nonpregnant females and females mated but not littered was excluded from statistical analysis.

The statistical analyses were followed to the parameters:
Body weight, Change in body weight, Feed consumption, Copulatory interval, Gestation length, Hematology, Urinalysis, Clinical chemistry, Absolute organ weights, Relative organ weights were analysed by Oneway ANOVA with Dunnett’s posttest for main groups and t-test for recovery groups (Parametric).

FOB ( Body temperature, Defecation, Urination, Rearing, Grip strength, Movements), Mean litter weights, Mean pup weight, Pre implantation loss, Pre natal loss, Post natal loss,Total No. of resorptions/dam, Total No. of early/late resorptions/dam were analysed by Oneway ANOVA with Dunnett’s posttest (Parametric).

Dams with live young born, with early/late resorptions, Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Litter size, Pup weight were analysed by KruskalWallis followed by the MannWhitney test.

Pregnancy rate, No. of live pups, No. of dead pups, No. of early resorptions, No. of late resorptions, No. of litters with/without dead pups, No. of litterswith/without resorptions were analysed by Chisquare test/ Fischer's Exact Test (Cross Tabs)

Reproductive indices:

Male fertility index (%): G1 83.3; G2 91.7; G3 75.0; G4: 100.0
Female fertility index (%): G1 91.7; G2 91.7; G3 91.7; G4: 100.0

Offspring viability indices:

Live birth index (%): G1 96.6; G2 92.7; G3 96.3; G4: 95.6
Day 4 survival index (%): G1 99.4; G2 99.3; G3 99.4; G4: 99.2

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in haematology at any of the dose tested in both the sex. However, statistical significant increase in percent neutrophils, and statistical significant decrease in percent lymphocytes in G2 (males), statistical significant decrease in total leucocyte count in G4R (females), statistical significant decrease in prothrombin time in G4R (recovery males), statistical significant decrease in absolute lymphocytes, monocytes, eosinophils and basophils in G4R (recovery females) was noted when compared with vehicle control group. These changes were considered incidental as the values were within historical range or due to random biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in clinical chemistry at any of the dose tested in both the sex.
However, statistical significant decrease in total protein and calcium levels in G3 (females) and statistical significant decrease in alkaline phosphatase and potassium levels in G4R (recovery females) were noted when compared with vehicle control group. These changes were considered incidental as the values were within historical range or due to random biological variation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): There were no clinical signs of toxicity and mortality/morbidity observed at any of the doses tested in both the sex

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There were no treatment related changes in mean body weight and percent change inbody weight with respect to day 1 at any of the doses tested in both the sex.

However, statistical significant decrease in percent change in body weight with respect to Day 1 on Day 7 in G2 (females) and G4 (females) and statistical significant decrease in percent change in body weight with respect to Day 1 on Day 7 in G4R (males) was noted when compared with vehicle control group. These changes are considered incidental due to lack of dose dependency and also the mean body weights were comparable.

There were no treatment related changes noted in feed consumption at any of the doses tested in both the sex.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No treatment related changes were noted.

ORGAN WEIGHTS (PARENTAL ANIMALS): There were no treatment related changes observed in the absolute and relative organ weights at any of the dose tested in both the sex.

However, statistical significant decrease in fasting body weight in G2 (females), statistical significant increase (slight) in relative testes weight in G4 (males), statistical significant increase in relative brain weight in G2 (females) and statistical significant increase in relative heart weight G4R (males) were noted whencompared with vehicle control group. All the observed changes were considered incidental and not treatment related because of the lack of dose dependency and/or there were no associated macroscopic and microscopic abnormalities noted.

GROSS PATHOLOGY (PARENTAL ANIMALS): There were no gross pathological changes (both external and internal) observed at any of the doses tested in both the sex.

HISTOPATHOLOGY (PARENTAL ANIMALS): No treatment related histopathological findings noticed in the study.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adeverse effects observed up to limit dose

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

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:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING): No treatment related effects were noted.

CLINICAL SIGNS (OFFSPRING): No clinical signs were noted.

BODY WEIGHT (OFFSPRING): No treatment related effects were noted.

GROSS PATHOLOGY (OFFSPRING): No treatment related effects were noted.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adeverse effects observed up to limit dose

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

OBSERVATIONS	Group & Dose (mg/kg body weight)
	G1 & 0	G2 & 100	G3 & 300	G4 & 1000
Pairs started (N)	12	12	12	12
No. of males impregnating the females	10	11	9	12
Male fertility index (%)	83.3	91.7	75.0	100.0
Females showing evidence of copulation (N)	12	12	12	12
Females achieving pregnancy (N)	11	11	11	12
Female fertility index (%)	91.7	91.7	91.7	100.0
Female fecundity index (%)	91.7	91.7	91.7	100.0
Parturition (%)	91.7	91.7	91.7	100.0
Conceiving days 1 - 5 (N)	10	8	7	10
Conceiving days 6 -20 (N)	2	4	5	2
Pregnancy = 21 days (N)	0	0	0	0
Pregnancy = 22 days (N)	10	11	8	8
Pregnancy = 23 days (N)	1	0	3	2
Pregnancy ˃ 23 days (N)	0	0	0	2
Dams with live young born (N)	11	11	11	12
Dams with live young at day 4 pp (N)	11	11	11	12
Corpora lutea/dam (mean)	12.5	11.6	10.9	11.8
Implants/dam (mean)	12.4	11.5	10.6	11.6
Live pups/dam at birth (mean)	11.8	10.4	9.9	10.8
Male Live pups/dam at birth (mean)	7.00	5.73	5.36	5.92
Female Live pups/dam at birth (mean)	4.82	4.64	4.55	4.92
Live pups/dam at day 4 (mean)	11.7	10.3	9.8	10.8
Male Live pups/dam at day 4 (mean)	6.91	5.64	5.27	5.83
Female Live pups/dam at day 4 (mean)	4.82	4.64	4.55	4.92
Live birth index (%)	96.62	92.74	96.33	95.75
Sex ratio (m/f) at birth (mean)	1.61	1.59	1.38	1.39
Male Litter weight at birth (mean)	36.85	32.83	29.38	34.4
Female Litter weight at birth (mean)	26.32	25.31	23.55	25.81
Litter weight at birth (mean)	65.16	58.14	52.93	60.21
Male Litter weight at day 4 (mean)	49.04	41.16	39.36	45.25
Female Litter weight at day 4 (mean)	32.97	31.77	29.59	34.44
Litter weight at day 4 (mean)	82.01	72.93	68.95	79.69
Male Pup weight at birth (mean)	5.57	5.64	5.47	5.85
Female Pup weight at birth (mean)	5.46	5.39	5.22	5.25
Pup weight at birth (mean)	5.65	5.58	5.35	5.6
Male Pup weight at day 4 (mean)	7.34	7.27	7.60	7.81
Female Pup weight at day 4 (mean)	6.93	6.83	6.68	7.01
Pup weight at day 4 (mean)	7.18	6.99	7.31	7.47
Day 4 survival index (%)	99.39	99.30	99.39	99.24

LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Females with 0	10	9	8	10
Females with 1	1	2	3	2
Pre-natal (implantations minus live births)
Females with 0	7	6	7	5
Females with 1	3	2	1	5
Females with 2	0	1	3	2
Females with >3	1	2	0	0
Post-natal (live births minus alive at post natal day 4)
Females with 0	10	10	10	11
Females with 1	1	1	1	1

pp: post partum

Conclusions:

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Hordaphos CC MIS was found to be 1000 mg/kg body weight when administerd to the main group males for two weeks pre-mating, during mating and up to the day before sacrifice during post- mating period (total of 35 days), the main group females treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day and the recovery group animals till first scheduled sacrifice of dam(s) (i.e. 41 days) by oral (gavage) to Sprague Dawley rats under the experimental conditions and the doses employed.

For reproductive and developmental toxicity assessment, the No Observed Adverse Effect Level (NOAEL) of the test item Hordaphos CC MIS was found to be 1000 mg/kg body weight, based on no adverse effects on the parameters like copulatory interval, gestation length, gestation/lactationbody weight, gestation/lactation feed consumption, litter weights, mean pup weights, uteri observations and no gross pathological findings in both dams and pups.

Executive summary:

The test item, Hordaphos CC MIS wasevaluated for possible adverse effects following repeated oral dosing for relatively limited period of time and to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to main and recovery groups. Each main group (G1, G2, G3and G4) consisted of 12 males and 12 females and recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R groups were administered with vehicle [Distilled water], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered at the dose volume of 10 mL/kg body weight.

The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 4 after which the pups were sacrificed on lactation Day 4 and females (dams) were sacrificed on lactation day 5 after overnight fasting (water allowed).

The recovery group animals were treated till first scheduled sacrifice of dam(s) and observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

The test item formulations at the concentrations 10 mg/mL (low dose) and 100 mg/mL (high dose) were stable up to 6 hours at room temperature as per the in-house stability study results (Bioneeds Study No.: BIO-ANM 116). However, freshly prepared test item formulations were administered to the animals.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination was carried out once before treatment for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice)for vehicle control and high dose main group animals andduring last weekfor recovery group animals. Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice). The Neurological/Functional examination was performed for recovery groups during the last week of the recovery period.

The clinical pathology (haematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) and for all recovery group animals. Urinalysis was performed from the five randomly selected males of each group during the last week of the treatment period for main groups and during last week of recovery period for recovery groups.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. Body weights of male and female pups were recorded separately on lactation days 1 and 4.

Gross pathology and organ weighing were performed on day 36 for main group males, on lactation day 4 for pups, lactation day 5 for dams and at end of recovery period for recovery group males and females. The number of corpora lutea, implantation sites and resorptions for dams were recorded.

Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure).

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortalityor morbiditywasobserved throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis, organ weights (both absolute and relative) were observed in both the sex.

The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights.

The animals (main group and recovery) did not reveal any treatment related gross as well as histopathological changes on examination.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

There is no evidence to suggest that a classification for reproductive toxicity is appropriate.

With reference to the OECD 422 study performed with, Hordaphos CC MIS, the structural analoge of the registration substance, it is concluded that the test item is not subject to classification and labelling according to Regulation 1272/2008/EC regarding reproductive toxicity.