Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant OECD 415 guideline study
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: (P) males ca. 6 weeks, females ca. 10 weeks
- Weight at study initiation: (P) Males: 191-255 g; Females: 211-271 g
- Housing: initially, in groups of 4 in polypropylene cages with stainless steel grid floors and tops. During the mating, animals were housed in similar cages on 1:1 male:female basis. After mating males were transferred to the original cages; females were housed individually during gestation and lactation in polypropylene cages with solid floors and stainless steel lids.
- Diet: pelleted diet (Rodent PMI 5002 (certified) diet, ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15/hour
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: formulations were prepared weekly

VEHICLE
- Concentration in vehicle: 0, 6.25, 25 and 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 21 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 1 of pregnancy
- After successful mating each pregnant female was caged individually during gestation and lactation in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in arachis oil was determined by gas chromatography using an external standard technique.
Duration of treatment / exposure:
76 days pre-mating, maximal 21 days mating, males were killed and examined upon evidence of successful mating, females and offspring were killed and examined on day 21 post-partum. Non-pregnant females were killed and examined after day 25 post-coitum.
Frequency of treatment:
Daily (except for females during littering/parturition)
Details on study schedule:
Age at mating of the mated animals in the study: mating was performed on day 76 of treatment.
Remarks:
Doses / Concentrations:
0, 25, 100 and 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 14-day dose-range finding study
- Rationale for animal assignment (if not random): the animals were allocated to dose groups using a randomisation procedure based on stratified body weights.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before and after the dosing, and 1 and 5 hours post-dosing during the working week. Animals were observed immediately before and after dosing and 1 hour post-dosing at the weekends or public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, then weekly for males until termination. Females were weighed weekly during maturation and daily during mating. Once mating was evident, body weights were recorded on days 1, 4, 7, 14 and 21 of post-coitum and post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
- Time schedule for examinations: During the maturation period, weekly food consumption was recorded for each cage. For females showing evidence of mating, food consumption was recorded for the period covering days 1-7, 7-14 and 14-21 post-coitum. For females with live litters, food consumption was recorded for the period covering days 1-7, 7-14 and 14-21 post-partum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt change.
Oestrous cyclicity (parental animals):
During mating, a vaginal smear was prepared for each female daily and the stage of the estrous cycle was recorded.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, numbers of homogenisation resistant spermatids, sperm motility, sperm morphology
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, the detachment and unfolding of pinna, incisor eruption and eyelid separation, reflexological response to stimuli by assessing surface righting reflex on Day 1 post partum and air righting reflex on Day 17 post-partum. Pupillary reflex and auditory startle response were performed on day 21 post-partum.

GROSS EXAMINATION OF DEAD PUPS
For external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals; after succesful mating.
- Maternal animals: All surviving animals; on day 21 post-partum; for non-pregnant females on or after day 25 post-coitum.

GROSS NECROPSY
- Gross necropsy consisted of full external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively: cervix, coagulating gland, epididymides, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus, vagina.
Postmortem examinations (offspring):
SACRIFICE
The off-spring was sacrificed on day 21 after birth.

GROSS NECROPSY
Gross necropsy consisted of full external and internal examinations.
Statistics:
Linear regression analysis, followed by ANOVA incorporating Levene's test for homogeneity of variance was used for data on organ weights, weekly body weight, litter weights, offspring body weights. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances, the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio, litter size and landmark developmental markers.
Chi-squared analysis was used for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance was used for the comparison of severity grades for the more frequently observed graded conditions.
Reproductive indices:
The following indices were calculated:
Mating index = (number of animals mated/number of animals paired) x 100%
Pregnancy index = (number of pregnant females/number of animals mated) x 100%
Parturition index = (number of females delivering live offspring/number of pregnant females) x 100%
Offspring viability indices:
The following indices were calculated:
Live birth index = (number of offspring alive on day 1/number of offspring born) x 100%
Viability index 1 = (number of offspring alive on day 4/number of offspring alive on day 1) x 100%
Viability index 2 = (number of offspring alive on day 7/number of offspring alive on day 4) x 100%
Viability index 3 = (number of offspring alive on day 14/number of offspring alive on day 7) x 100%
Viability index 4 = (number of offspring alive on day 21/number of offspring alive on day 14) x 100%
Viability index 5 = (number of offspring alive on day 21/number of offspring alive on day 1) x 100%
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See discussion and repeated dose toxicity section 7.5.1
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See discussion and repeated dose toxicity section 7.5.1
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See discussion and repeated dose toxicity section 7.5.1
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Description (incidence and severity):
Test substance intake: Test substance is administered as gavage
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): one male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during partutition. Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): body weight gain for males of 500 mg/kg bw/day group was generally lower (average -22%, range -9% - -43%) than control animals throughout much of the treatment period, with statistical differences observed in Weeks 4, 5, 6 and 10.

FOOD CONSUMPTION (PARENTAL ANIMALS): females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21%, range -17% - -28%) throughout lactation, with statistically significant differences throughout 3 weeks.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): there were no treatment-related effets on female estrous cycles or on the type or proportion of females with anomalous estrous cycle.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): there were no toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid count.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): mating performance was good in all groups with the majority of animals mating within the first four days of pairing. Subsequent pregnancy rate was unaffected by treatment with only 1, 1, 1 and 2 females failing to achieve pregnancy in the control, 25, 100 and 500 mg/kg bw/day groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS): there were no treatment-related effects.

GROSS PATHOLOGY (PARENTAL ANIMALS): the adult male treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gasto-intestinal tract. The female treated with 500 mg/kg bw/day that was found dead around partutition had 21 foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive fetuses in-utero and red/brown staining aroudn the ano-genital region and dark contents in the stomach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach.

HISTOPATHOLOGY (PARENTAL ANIMALS): no treatment-related microscopic changes were observed in the parental animals.
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on fertility were noted at the highest tested dose.
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Repeated dose toxicity was seen at 100 mg/kg bw based on gestation length increase.
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
effects observed, treatment-related
VIABILITY (OFFSPRING): Life birth index for 500 mg/kg bw/day females was significantly lower (-15%) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and day 1.

CLINICAL SIGNS (OFFSPRING): Skin sloughing was detected in offspring during the first week of lactation in all treatment groups (more pronounced in the high dose group) together with multiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters.
A delay in the onset (+ 2.1 days) and completion ( + 2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning.

BODY WEIGHT (OFFSPRING): Body weight gain at 500 mg/kg bw/day was lower (-58%) than control animals for the first week of age and again from day 14 to weaning (day 21 of age) (-17%). Litter weight, at this treatment group, was notably lower than control animals throughout lactation.

ORGAN WEIGHTS (OFFSPRING): No treatment-related changes were detected.

GROSS PATHOLOGY (OFFSPRING): Offspring from females treated with 500 and 100 mg/kg bw/day showed skin sloughing at necropsy. Offspring from females treated with 500 mg/kg bw/day also showed sparse fur coverage.

HISTOPATHOLOGY (OFFSPRING): Acanthosis and hyperkeratosis were seen in relation to treatment for the skin of male and female F1 generation animals treated with 500 and 100 mg/kg bw/day.
Reproductive effects observed:
not specified
Conclusions:
In the one-generation study with rats, the NOAEL for parental toxicity was set at 25 mg/kg bw/day, based on gestation length increase in the 100 and 500 mg/kg bw dose groups. The NOAEL for developmental effects was set at 25 mg/kg bw/day, based on the skin effects (skin peeling and flaking, and acanthosis and hyperkeratosis observed at necropsy) in male and female F1 animals at 100 mg/kg bw.
Executive summary:

Reproductive toxicity of Lyral was studied in a GLP-compliant study with Sprague-Dawley rats, conducted according to OECD Guideline 415. The substance was administered at dose levels of 0, 25, 100 and 500 mg/kg bw/day as a solution in arachis oil to groups of 24 rats/sex/dose for 76 days pre-mating and during maximum 21 days mating, after which males were killed, while females and subsequent offspring were killed and examined on day 21 post-partum. Non-pregnant females were killed and examined after day 25 post-coitum. One male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during parturition. Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation. Body weight gain for males of the 500 mg/kg bw/day group was generally lower (average -22%, range -9% - -43%) than control animals throughout much of the treatment period, with statistical differences observed in weeks 4, 5, 6 and 10. Females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21%, range -17% - -28%) throughout lactation, with statistically significant differences throughout 3 weeks. There were no treatment-related effects on female estrous cycles or on the type or proportion of females with an anomalous estrous cycle, or toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts. Mating performance was good in all groups with the majority of animals mating within the first four days of pairing. Subsequent pregnancy rate was unaffected by treatment with only 1, 1, 1 and 2 females failing to achieve pregnancy in the control, 25, 100 and 500 mg/kg bw/day groups, respectively. An increased gestation length was observed in the 100 and 500 mg/kg bw dose groups. At necropsy, the adult male treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gasto-intestinal tract. The female treated with 500 mg/kg bw/day that was found dead around parturition had 21 foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive fetuses in-utero and red/brown staining around the ano-genital region and dark contents in the stomach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach. No treatment-related microscopic changes were observed in the parental animals. Based on the observed increased gestation length, the NOAEL for parental toxicity was set at 25 mg/kg bw/day.

Life birth index for 500 mg/kg bw/day females was significantly lower (-15%) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and day 1. Skin sloughing was detected in offspring during the first week of lactation in all treatment groups (more pronounced in the high dose group) together with multiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters. A delay in the onset (+ 2.1 days) and completion (+ 2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning. Body weight gain in offspring at 500 mg/kg bw/day was lower (-58%) than in control animals for the first week of age and again from day 14 to weaning (day 21 of age) (-17%). Litter weight, at this treatment group, was notably lower than control animals throughout lactation. Offspring from females treated with 500 and 100 mg/kg bw/day showed skin sloughing at necropsy. Offspring from females treated with 500 mg/kg bw/day also showed sparse fur coverage. Acanthosis and hyperkeratosis were seen in relation to treatment for the skin of male and female F1 generation animals treated with 500 and 100 mg/kg bw/day. Based on the skin effects in F1 offspring, the NOAEL was set at 25 mg/kg bw/day for the F1 generation.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The selected study is a GLP compliant guideline study and has klimisch score of 1.
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 fertility ot the substance was assessed in a one-generation study.

One-generation study: Reproductive toxicity of Lyral was studied in a GLP-compliant study with Sprague-Dawley rats, conducted according to OECD Guideline 415. The substance was administered at dose levels of 0, 25, 100 and 500 mg/kg bw/day as a solution in arachis oil to groups of 24 rats/sex/dose for 76 days pre-mating and during maximum 21 days mating, after which males were killed, while females and subsequent offspring were killed and examined on day 21 post-partum. Non-pregnant females were killed and examined after day 25 post-coitum.

Fertility: There were no treatment-related effects on female estrous cycles or on the type or proportion of females with an anomalous estrous cycle, or toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts. Mating performance was good in all groups with the majority of animals mating within the first four days of pairing. Subsequent pregnancy rate was unaffected by treatment with only 1, 1, 1 and 2 females failing to achieve pregnancy in the control, 25, 100 and 500 mg/kg bw/day groups, respectively.

Parental effects: One male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during parturition. Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation. Body weight gain for males of the 500 mg/kg bw/day group was generally lower (average -22%, range -9% - -43%) than control animals throughout much of the treatment period, with statistical differences observed in weeks 4, 5, 6 and 10. Females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21%, range -17% - -28%) throughout lactation, with statistically significant differences throughout 3 weeks.

An increased gestation length was observed in the 100 and 500 mg/kg bw dose groups. At necropsy, the adult male treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gastro-intestinal tract. The female treated with 500 mg/kg bw/day that was found dead around parturition had 21 foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive fetuses in-utero and red/brown staining around the ano-genital region and dark contents in the stomach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach. No treatment-related microscopic changes were observed in the parental animals. Based on the observed increased gestation length, the NOAEL for parental toxicity was set at 25 mg/kg bw/day.

Developmental toxicity is discussed below.


Short description of key information:
In the one-generation study with rats, the NOAEL for fertility has been set to 500 mg/kg bw, the highest dose tested. The NOAEL for developmental effects was set at 25 mg/kg bw/day, based on the delays in the development of the pups (delay in the pinna unfolding) and the skin effects (skin peeling and flaking, and acanthosis and hyperkeratosis observed at necropsy) in male and female F1 animals. The parental toxicity was set at 25 mg/kg bw/day, based on gestation length increase in the 100 and 500 mg/kg bw dose groups.

Justification for selection of Effect on fertility via oral route:
The fertility endpoint needs to be assessed for the REACH regulation. The one-generation study according to OECD TG 415 fulfills this information

Effects on developmental toxicity

Description of key information
The key study for developmental toxicity is the one-generation study. In this study a NOAEL of 25 mg/kg bw is derived. At higher doses in this study effect on delayed development of the pups were seen and on effects on skin, such as skin peeling were observed. Because of these latter effects are rare two additional studies were performed, which did not lead to a change in NOAEL. 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The well conducted GLP-compliant research study which is a follow up study of the OECD 415 study is reliable is needed to assess the effects on pups via lactation but does not include all developmental toxicity parameters
Principles of method if other than guideline:
Oral (gavage) repeated-dose toxicity study in adult rats with determination of the test substance in breast milk, including neonatal evaluation and recovery period.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Weight at study initiation: 298-368 g
- Housing: individually in nesting boxes. Each dam and delivered litter was housed in a common nesting box during the postpartum period. After weaning, the F1 generation rats were individually housed in stainless steel wire-bottom cages.
- Diet: Certified Rodent Diet # 5002, ad libitum
- Water: local water that has been processed by passage through a reverse osmosis membrane, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): at least 10/hr
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions of the test article in the vehicle were prepared at the testing facility on a weekly basis and aliquotted for daily use. Prepared formulations were stirred continuously using a magnetic stir bar and stir plate during sample collection, aliquotting and dosage administration. Prepared formulations were stored refrigerated (2-8 ºC) and protected from light. Daily aliquotes were mixed thoroughly using a magnetic stir bar and stir plate for at least 30 min prior to dosage administration.

VEHICLE
- Concentration in vehicle: 0, 2.5, 6.25 and 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): 1237573
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose formulations were analyzed for HMPCC by gas chromatography with flame ionization detection (GC-FID). The method was validated for the analysis of dose formulations at concentrations ranging from 1.9 to 125 mg/mL of HMPCC in peanut oil.
Details on mating procedure:
- Impregnation procedure: cohoused
- Verification of same strain and source of both sexes: yes
- Length of cohabitation: 1-2 consecutive days
- Proof of pregnancy: referred to as day 0 of pregnancy
The female rats were naturally bred at the Supplier before shipment to the Testing Facility. The rats were shipped to the Testing Facility after the last mating day to arrive on DG 15 or 16. Rats were placed into nesting boxes in consecutive order, by day of gestation on the day of arrival.
Duration of treatment / exposure:
Female F0 rats: from day 1 postpartum through day 21 postpartum. F1 generation pups were not directly given the test article, but might have been exposed to the test article via maternal milk during the lactation period.
Frequency of treatment:
Daily
Duration of test:
F0 rats: until day 15 (5 dams/dose) or 22 (5 dams/dose) postpartum
F1 rats: until day 15 postpartum or until days 64-67 postpartum
Remarks:
Doses / Concentrations:
0 (vehicle), 10, 25 and 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on previously conducted one-generation study and repeated dose toxicity study with neonatal evaluation. The 500 mg/kg bw/day dose was selected because it is the dosage at which effects were observed in both the dams and the offspring. The 25 mg/kg bw/day dosage was selected because it is the developmental NOAEL. The 10 mg/kg bw/day dosage was selected because it is the expected NOEL.
- Rationale for animal assignment: based on computer-generated (weight-ordered) randomization procedure
- Other: Groups of 10 female rats were administered the test article at dose levels of 10, 25 and 500 mg/kg bw/day or the vehicle (arachis oil) orally (via gavage) once daily on days 1 through 21 postpartum. F0 generation dams were sacrificed on either days postpartum (DP) 15 (five dams per group and their respective litters) or DP 22 (five dams per group). F1 generation rats not sacrificed on DP 15 were weaned on DP 22 and observed until sacrifice upon completion of the postweaning recovery period (six to seven weeks after weaning, DPs 64 to 67). The following parameters were evaluated: viability, clinical observations, body weights and body weight gains, feed consumption, natural delivery observations, necropsy observations. Maternal milk, blood and urine were collected.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all F0 generation rats were observed for viability at least twice daily. These rats were also examined for clinical observations and general appearance daily beginning the day after arrival during the acclimation period. Observations for clinical signs of effects of the test article were also made daily before dosage and between one and two hours after dosage administration, at the end of the normal working day and prior to sacrifice. Maternal behaviour was also observed on days postpartum (DPs) 1, 4, 7, 14, 18 and 22.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded daily beginning the day after arrival and prior to sacrifice on DP 15 or 22.

FOOD CONSUMPTION: Yes
Feed consumption was recorded daily beginning the day after arrival during the acclimation period and DPs 1, 4, 7, 10 and 14 during the dosage period. Feed consumption was not tabulated after DP 14, when it was expected that pups began to consume maternal feed.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 15 or 22
- Organs examined: thoracic, abdominal and pelvic viscera.

OTHER: Urine samples were collected overnight for evaluation from the first 5 dams per dosage group on DP 14. These 5 dams were fasted overnight for approximately 14 hours. Milk samples (0.1 mL to 0.2 mL) were collected from each dam on DP 14 and all remaining dams on DP 21 at approximately four to six hours postdosage. Whole blood samples were collected from each dam on DP 14 and all remaining dams on DP 21 at approximately four to six hours postdosage and after milk collection.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- number of offspring per litter (live and dead pups)
- number of implantation sites
- general condition of dam and litter during the postpartum period
- viability indices (percentage of pups born that survive 4 and 7 days)
- lactation index (percentage of pups born that survive 22 days).
Statistics:
All data were tabulated, summarized and statistically analysed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft® Excel [part of Microsoft Office 97/2000/2003/XP], Quattro Pro 8 and the SAS System (version 6.12). Averages and percentages were calculated. Litter values were used where appropriate. Additional procedures and/or analyses were performed, if appropriate.
Clinical observations and other proportional data were analysed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g. maternal body weights, body weight changes, feed consumption values and litter averages for per cent male foetuses, per cent resorbed conceptuses, foetal body weights and foetal anomaly data) were analysed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test is not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal Wallis Test11 was used (≤75% ties). In cases where the Kruskal Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyse the data.
Count data was evaluated using the procedures described above for the Kruskal-Wallis Test.
Indices:
Viability index: percentage of pups born that survive 4 and 7 days
Lactation index: percentage of pups born that survive 22 days
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal deaths were attributed to treatment of lactating female rats with any dosage of Lyral. All clinical and necropsy observations were considered unrelated to Lyral. Body weight gains for the first two weeks of the lactation period were comparable among the four dosage groups. At 500 mg/kg bw/day, body weight gains for the five remaining rats assigned to scheduled sacrifice on DP 22 were increased on DPs 14 to 22 and overall for DPs 1 to 22, as compared to the vehicle control group values. Absolute and relative feed consumption values in the 500 mg/kg bw/day dosage group were significantly lower than the corresponding vehicle control group value on DPs 1 to 14 (13% lower than the vehicle control group value).
Breast milk samples were collected from all rats on DP 14 (10 per dosage group) and the surviving rats on DP 21 (four or five per dosage group) and analyzed for Lyral levels. No quantifiable levels of Lyral were detected in any rats from the 0 (Vehicle), 10, or 25 mg/kg bw/day dosage groups on either DP 14 or 21. For the 500 mg/kg bw/day dosage group, on DP 14 two of 10 rats had quantifiable levels of Lyral in the milk, with an average concentration of 73.5 ng/mL. The number of rats with detectable peaks for Lyral which fell below the limit of quantitation (50 ng/mL) on DP 14 was increased in the 500 mg/kg bw/day dosage group in comparison to the other dosage groups. On DP 21, four of five rats from the 500 mg/kg bw/day dosage group had quantifiable levels of Lyral in the milk, with an average concentration of 59.85 ng/mL.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
F1 generation pup body weights were reduced at 500 mg/kg bw/day during the lactation period, specifically on DPs 4 through 21. The average pup body weight on DP 22 was 83% of the vehicle control group value in the 500 mg/kg bw/day dosage group. The difference in pup body weight relative to the vehicle control group appeared to be greater in the female pups than in the male pups. In addition, female pup body weights in the 500 mg/kg bw/day dosage group were significantly reduced on DP 14, as compared to the vehicle control group value. Clinical signs that were observed in the F1 generation during the lactation period and attributed to maternal treatment with 500 mg/kg bw/day of Lyral included skin peeling and cold to touch. Skin peeling occurred in 109 of 111 surviving pups in the 500 mg/kg bw/day dosage group. This clinical sign was first observed as early as DP 8, and it persisted to necropsy on DP 15 or through weaning and into the postweaning recovery period. Skin peeling in the 500 mg/kg bw/day dosage group was associated with Lyral in the milk. No skin peeling was observed at 0 (Vehicle), 10 or 25 mg/kg bw/day and there was no Lyral detected in the milk at these dosages. No significant necropsy observations were observed in any of the F1 generation pups in any dosage group from dams selected for sacrifice on DP 15.
Abnormalities:
not specified
Developmental effects observed:
not specified

Post-weaning period

Twenty-one, 32, 21 and 22 male rats from the 0, 10, 25 and 500 mg/kg bw/day maternal dosage groups, respectively, and 28, 31, 20 and 36 female rats from the 0, 10, 25 and 500 mg/kg bw/day maternal dosage groups, respectively, were weaned on DP 22 for evaluation during the postweaning period.

Five F1 male rats (1, 1 and 3 from the 10, 25 and 500 mg/kg bw/day maternal dosage groups, respectively) were humanely euthanized on DPs 23, 24 or 59 because of adverse clinical signs or a broken palate. In addition, one F1 female rat in the 10 mg/kg bw/day maternal dosage group was also humanely euthanized on DP 32 because of a limb injury. Three F1 female rats in the 500 mg/kg/day maternal dosage group were found dead on DPs 24 or 27. Four of the deaths were considered unrelated to treatment [the male rat with the undetermined cause of death (10 mg/kg bw/day), the male rat with the broken palate (25 mg/kg bw/day), the female rat with the fractured femur (10 mg/kg bw/day) and the female rat with the suspected urinary tract infection (500 mg/kg bw/day) that had gross lesions consistent with a urinary tract infection]. Five of the deaths (i.e., unscheduled or found dead) at 500 mg/kg bw/day were attributed to a failure to thrive and were considered an effect of maternal treatment with the test article. All other F1 generation rats survived to scheduled sacrifice on DPs 64 to 67.

Clinical signs observed in the F1 rats during the postweaning period that were attributed to maternal treatment with 500 mg/kg bw/day of Lyral included mild dehydration (based on skin turgor) in both sexes, urine-stained abdominal fur in the F1 female rats and skin peeling in both sexes. Each of these clinical signs occurred in significantly more rats in the 500 mg/kg bw/day maternal dosage group than in the corresponding vehicle control groups. Eleven of 22 F1 male rats and all 36 F1 female rats at 500 mg/kg bw/day had peeling skin during the postweaning period. This effect was observed on DPs 23 (i.e., the first day of observation after weaning on DP 22), 24, 25 or 26, and resolved in both sexes by DP 35, two weeks into the postweaning recovery period. No skin peeling or other clinical signs were observed at 10 or 25 mg/kg bw/day Lyral.

All necropsy observations in the F1 generation male and female rats were considered unrelated to maternal treatment with Lyral.

Reflecting reductions in body weights that occurred prior to weaning, F1 male and female rats in the 500 mg/kg bw/day maternal dosage group weighed significantly less than the vehicle control group rats on DPs 23, 30, 37, 44 and 51 (males only), reflecting reductions in body weights that occurred prior to weaning. Corresponding significant reductions in body weight gains occurred in the F1 male rats at 500 mg/kg bw/day on DPs 23 to 30 and DPs 30 to 37, in comparison to the vehicle control group values. Thereafter, body weights and body weight gains were, in general, comparable among the dosage groups. Terminal body weights for the F1 male and female rats were comparable among the dosage groups.

In F1 male rats, statistically significant increases in relative feed consumption occurred in the 500 mg/kg bw/day maternal dosage group at each tabulated interval within the postweaning period (DPs 23 to 64), as compared to the vehicle control group values. Similar observations occurred in the F1 female rats, with the exception of DPs 58 to 64. These changes in relative feed consumption were considered related to maternal treatment with HMPCC because the smaller rats in this group ate approximately the same amount of feed as the slightly larger male or female rats in the other dosage groups resulting in an average increase in relative feed consumption.

Conclusions:
Administration of Lyral by gavage to female rats during lactation period (post-partum days 1-21) resulted in mortality, reductions in pup body weight and observations of skin peeling in the F1 pups at a dose level of 500 mg/kg bw/day. No effects were observed at dose levels of 10 and 25 mg/kg bw/day. Lyral could be detected in breast milk in the 500 mg/kg bw/day group at quantifiable levels. The skin peeling effects in F1 generation pups disappeared by post-partum day 35, 2 weeks into the post-weaning recovery period. The NOAEL for developmental toxicity was set at 25 mg/kg bw/day. The NOAEL for maternal toxicity was considered to correspond to 500 mg/kg bw/day as the observed changes (reduced food consumption and increased body weight) were considered to be non-adverse.
Executive summary:

Introduction: In order to determine if the effects of skin peeling observed in the offspring in earlier studies were related to the test material transmission through maternal milk and if so, whether the effect was reversible following weaning, a GLP-compliant study was conducted.

Methods: In the present study groups of 10 pregnant female rats were allowed to deliver litters and then administered the test substance at dose levels of 0, 10, 25 and 500 mg/kg bw/day on days 1 -15 or 1 -22 post-partum. The concentrations of the test substance in breast milk were evaluated on post-partum days 14 and 21. The pups were allowed to wean and observed for 64 to 67 days post-partum to determine whether skin effects observed in two earlier studies were reversible.

Results: Maternal effects: Lyral at 500 mg/kg bw/day caused transient reductions in maternal body weight gain early on during the lactation treatment period. At 500 mg/kg bw/day, maternal body weight gains at the end of the lactation treatment period (days postpartum (DPs) 14 to 22) were increased relative to the vehicle control group value, such that the overall body weight gains for the entire lactation period were 25% greater than the vehicle controls. Lyral also reduced maternal absolute and relative feed consumption values in the 500 mg/kg bw/day dosage group for the entire lactation period, as compared to vehicle controls. Lyral was quantifiable in the milk of two of 10 rats on DP 14 and four of five rats on DP 21 in the 500 mg/kg bw/day dosage group. No Lyral was detected in the milk of rats treated with 0 (Vehicle), 10 or 25 mg/kg bw/day Lyral on either DP 14 or 21.

Results F1: Observations in the F1 generation from dams treated with 500 mg/kg bw/day test substance during the lactation period included mortality, reductions in pup body weights and observations of skin peeling. The reductions in pup body weights continued into the postweaning period for both F1 male and female rats, but did not affect the terminal body weight or the cumulative body weight gains during the postweaning period. These reductions during the postweaning period reflected the effects of Lyral on pup body weights that occurred prior to weaning.

The observation of skin peeling occurred in all litters in the 500 mg/kg bw/day maternal dosage group as early as lactation Day (or Day of partitioning DP) 8 and, in general, persisted into the postweaning recovery period until recovery by DP 35. Skin peeling during the postweaning period occurred more frequently in the F1 female rats than in the F1 male rats. All 36 F1 female rats in the 500 mg/kg bw/day dosage group had the observation on one or more occasions during the postweaning period, while only 11 of 22 male rats had the observation during this same period. Skin peeling in the 500 mg/kg bw/day group was associated with Lyral in breast milk. The skin peeling resolved two weeks into the postweaning recovery period. No skin peeling was observed in 0 (vehicle), 10 and 25 mg/kg bw/day groups and there was no test substance detected in the milk at these dose levels. Transient, but increased incidences of cold to touch occurred in the pups prior to weaning, and mild dehydration occurred on one or more occasions after weaning in both F1 male and female rats. Several F1 female rats also had observations of urine-stained abdominal fur during the postweaning period.

The variation in body weight and food consumption may be related to systemic toxicity and therefore the 500 mg/kg bw is considered a LOAEL for the dams. Based on the results of the study, the NOAEL for viability and growth in the offspring was set at 25 mg/kg bw/day. Treatment of dams with 500 mg/kg bw/day Lyral during the lactation period resulted in quantifiable levels of Lyral in the milk which was associated with increased incidences of skin peeling in the F1 generation pups. The clinical observation of skin peeling resolved by post-partum day 31, 2 weeks into the postweaning recovery period and is therefore considered a reversible effect.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The selected study is a GLP compliant study and has klimisch score of 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

One-generation study, developmental effects

See for methods and results on repeated dose systemic toxicity the section on fertility above.

Life birth index for 500 mg/kg bw/day females was significantly lower (-15%) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and day 1. Skin sloughing was detected in offspring during the first week of lactation in all treatment groups (more pronounced in the high dose group) together with multiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters. A delay in the onset (+ 2.1 days) and completion (+ 2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning. Body weight gain in offspring at 500 mg/kg bw/day was lower (-58%) than in control animals for the first week of age and again from day 14 to weaning (day 21 of age) (-17%). Litter weight, at this treatment group, was notably lower than control animals throughout lactation. Offspring from females treated with 500 and 100 mg/kg bw/day showed skin sloughing at necropsy. Offspring from females treated with 500 mg/kg bw/day also showed sparse fur coverage. Acanthosis and hyperkeratosis were seen in relation to treatment for the skin of male and female F1 generation animals treated with 500 and 100 mg/kg bw/day. Based on the skin effects in F1 offspring, the NOAEL was set at 25 mg/kg bw/day for the F1 generation.

A new study was designed to determine the potential for adverse effects on the offspring observed in the one-generation reproductive study. It was designed to evaluate:

1) whether the pup dermal effects observed in the one-generation reproductive toxicity study were due to pre-or post-natal exposure to Lyral;

2) whether Lyral produced a functional zinc deficiency in the dams, thereby producing skin sloughing, peeling and/or flaking in pups. Fifty female rats were randomly assigned to 6 dosage groups:

 Dose groups  Doses in mg/kg bw  Number of rats  Dose administration
 Ia  0 (vehicle)  10  GD 0 through DG 21 or 24
 Ib  0 (vehicle)  10  LD 1 through 21
 II  500  10  GD throuhg DG 21 or 24
 III  500  10  LD 1 through LD 21
 IV  0 (vehicle)  5  GD 0 through 14
 V  500  5  GD 0 through 14

Results: Administration of Lyral at 500 mg/kg bw/day caused mortality as a result of dystocia and adverse clinical signs in the dams treated during the gestation period (Group II). Reductions in body weight gain occurred during the gestation treatment period; although not statistically significant, the reductions were substantial enough (90.3% of the concurrent vehicle control group value) to be toxicologically important. No apparent effects of Lyral on body weight gain were noted during the lactation period, following the cessation of test article administration. At sacrifice, the average terminal body weight was significantly increased, reflecting significant increases in maternal body weight gains after treatment was stopped. Serum chemistry parameters during early lactation, specifically glucose levels, blood urea nitrogen levels and alkaline phosphatase levels and the albumin to globulin ratio were significantly increased by 500 mg/kg bw/day Lyral (Group II). Treatment of the dams during the lactation period (Group III) resulted in increased liver weights, body weights (with fluctuating periods of body weight gain and body weight loss) and terminal weights. Cholesterol levels were lower in dams treated during the lactation period (Group III), while creatinine, aspartate aminotransferase, inorganic phosphate levels and albumin to globulin ratios were significantly increased in these dams. Treatment of dams with Lyral during gestation or lactation had no biologically important effect on zinc or metallothionein levels. Pups growth and viability was affected by Lyral treatment during the gestation period (Group II), in that increased incidences of stillbirths occurred as well as pup deaths near parturition and up to lactation day 3. As a result, the viability index was significantly reduced, the lactation index was reduced, and live litter sizes were significantly lower than control group values (Group Ia). Transient reductions in pup weights were noted in the F1 generation as a result of maternal treatment with Lyral during the gestation period (Group II). Clinical signs of cold to touch, often associated with pup mortality, and transient observations of flaking occurred in F1 generation pups. These effects were observed at a dose that caused reduced maternal body weight gains and maternal mortality. Observations in the F1 generation from dams treated during lactation (Group III) included significantly reduced viability and lactation indices, reductions in pup weights that were sustained until sacrifice, compared to the vehicle control (Group Ib), and persistent observations of skin peeling in all litters. In conclusion, when pregnant rats were treated with Lyral during the gestation period, transient clinical signs of flaking were noted in the F1 generation pups with relatively few observations of skin peeling. This effect occurred at maternally toxic levels. Conversely, treatment of the dams with Lyral during the lactation period resulted in skin peeling in all F1 generation pups, without resolution, and with minimal observations of flaking prior to sacrifice.

Study 2: In order to determine if the effects observed in the offspring in abovementioned study were related to the test material transmission through maternal milk and if so, whether the effect was reversible following weaning, another GLP-compliant study was conducted (Charles River Laboratories, 2009b).

Method: Groups groups of 10 pregnant female rats were allowed to deliver the litters and then administered the test substance at dose levels of 0, 10, 25 and 500 mg/kg bw/day on days 1-15 or 1-22 post-partum. The concentrations of the test substance in breast milk were evaluated on post-partum days 14 and 21. The pups were allowed to wean and observed for 64 to 67 days post-partum to determine whether skin effects observed in two earlier studies were reversible.

Results: Lyral at 500 mg/kg bw/day caused transient reductions in maternal body weight gain early on during the lactation treatment period. At 500 mg/kg bw/day, maternal body weight gains at the end of the lactation treatment period (days postpartum (DPs) 14 to 22) were increased relative to the vehicle control group value, such that the overall body weight gains for the entire lactation period were 25% greater than the vehicle controls. Lyral also reduced maternal absolute and relative feed consumption values in the 500 mg/kg bw/day dosage group for the entire lactation period, as compared to vehicle controls. Lyral was quantifiable in the milk of two of 10 rats on DP 14 and four of five rats on DP 21 in the 500 mg/kg bw/day dosage group. No Lyral was detected in the milk of rats treated with 0 (Vehicle), 10 or 25 mg/kg bw/day Lyral on either DP 14 or 21. The observed effects in maternal animals are considered to be adverse, the dose level of 500 mg/kg bw/day is considered to be a LOAEL for maternal toxicity. Results F1 generation: Observations in the F1 generation from dams treated with 500 mg/kg bw/day test substance during the lactation period included mortality, reductions in pup body weights and observations of skin peeling. The reductions in pup body weights continued into the postweaning period for both F1 male and female rats, but did not affect the terminal body weight or the cumulative body weight gains during the postweaning period. These reductions during the postweaning period reflected the effects of Lyral on pup body weights that occurred prior to weaning. The observation of skin peeling occurred in all litters in the 500 mg/kg bw/day maternal dosage group as early as DP 8 and, in general, persisted into the postweaning recovery period until recovery by DP 35. Skin peeling during the postweaning period occurred more frequently in the F1 female rats than in the F1 male rats. All 36 F1 female rats in the 500 mg/kg bw/day dosage group had the observation on one or more occasions during the postweaning period, while only 11 of 22 male rats had the observation during this same period. Skin peeling in the 500 mg/kg bw/day group was associated with Lyral in breast milk, indicating oral absorption and systemic exposure. The skin peeling resolved two weeks into the postweaning recovery period. No skin peeling was observed in 0 (vehicle), 10 and 25 mg/kg bw/day groups and there was no test substance detected in the milk at these dose levels. Transient, but increased incidences of cold to touch occurred in the pups prior to weaning, and mild dehydration occurred on one or more occasions after weaning in both F1 male and female rats. Several F1 female rats also had observations of urine-stained abdominal fur during the postweaning period. Based on the results of the study, the NOAEL for viability and growth in the offspring was set at 25 mg/kg bw/day. Treatment of dams with 500 mg/kg bw/day Lyral during the lactation period resulted in quantifiable levels of Lyral in the milk which was associated with increased incidences of skin peeling in the F1 generation pups. The clinical observation of skin peeling resolved by post-partum day 31, 2 weeks into the postweaning recovery period.

Summary developmental toxicity: Developmental toxicity has been assessed in several studies a one-generation study and two developmental toxicity studies. 1) The key study is the one-generation study according to OECD 415 (0, 25, 100 and 500 mg/kg bw) in which the NOAEL of 25 mg/kg bw was found. In this study the animals were administered Lyral during gestation and lactation (OECD 415); 2a) In a second limit dose study (0 and 500 mg/kg bw) the exposure was again during gestation and lactation; 2b) In the same study with additional animals exposure (limit dose) during lactation was performed. 3) A study where pups were exposed during lactation only (0, 10, 25, 500 mg/kg bw).

In a one-generation reproductive toxicity study with rats, life birth index was significant lower and 6 females had a total litter loss. At 500 mg/kg bw a delay in onset and completion of the pinna unfolding was evident and less so at 100 mg/kg bw. Also the number of offspring passing surface righting, air righting and pupil reflex was reduced. Body weight and body weight gain of the pups was lower throughout lactation. Skin sloughing was seen in the pups at 500 and less so in 100 mg/kg bw. This is a rare effect and therefore additional studies were performed to see whether the effects was more pronounced when dosed during gestation or during lactation. In a limit dose study, a control and a dose of 500 mg/kg bw was administered. Again at 500 mg/kg bw significant maternal and development effects were seen. Pups of dams treated during gestation showed, however, less skin effects compared to dams treated during lactation. Therefore, another study was performed at which dams were only exposed during lactation at 500, 25 and 10 mg/kg bw. Lyral was found in the milk at 500 mg/kg bw but not in the 25 and 10 mg/kg bw. The skin sloughing was seen seen in the 500 mg/kg bw and was reversible in the post weaning period at Day 35 of parturition. At this dose significant maternal toxicity was seen including liver toxicity possibly be indicative for overloading the metabolic pathway.

A NOAEL for parental toxicity of 25 mg/kg bw/day was established based on the OECD TG 415 being the key study for developmental toxicity, based on mortality,clinical signs, decreases in body weight gain and food consumption as well as liver effects at the highest dose of 500 mg/kg bw. In addition, at 500 mg/kg bw gestation length was increased and this wal also seen at 100 mg/kg bw but less pronounced. This gestation length increase at 100 mg/kg bw was finally the most critical effect on which base the NOAEL was set at 25 mg/kg bw. For the F1 generation, a NOAEL of 25 mg/kg bw/day was established based on delayed growth and on skin effects in male and female F1 animals. This NOAEL of 25 mg/kg bw was confirmed in the developmental toxicity in which the dams were exposure during lactation only.


Justification for selection of Effect on developmental toxicity: via oral route:
Developmental toxicity needs to be assessed for the REACH regulation. Several studies on this endpoint has been conducted. The developmental toxicity from the one-generation toxicity study has been used because a NOAEL on this endpoint has been derived.

Justification for classification or non-classification

Fertility: Based on the lack of adverse effects on fertility at the highest dose tested (500 mg/kg bw) in the one-generation study with rats, classification of Lyral for effects on fertility is not warranted in accordance with EU Directive 67/548 (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Developmental toxicity: In the one-generation reproductive toxicity study with rats, administration of 500 mg/kg bw/day to dams resulted in decreased live birth indices and reduced pup viability, as well as reduced pup body weight and body weight gain. At 100 mg/kg bw increase in gestation length in the dams was followed by a delay in pup development (pinna unfolding, incisor eruption, eye opening and reflex development). This delay was minimal at 100 mg/kg bw and more severe at 500 mg/kg bw. The presence of skin peeling, acanthosis, and hyperkeratosis in the offspring were evident at dose levels of 100 and 500 mg/kg bw/day. These doses caused minimal and significant maternal toxicity, respectively. In addtion, in two follow up studies these skin effects showed to be reversible during the postweaning period. Therefore classification and labelling is not warranted.

Developmental toxicity via lactation: Pups are affected during lactation when dams are exposed at 500 mg/kg bw during lactation only. Some mortality was seen, some failed to thrive and body weight was lower until weaning. Also the skin peeling effects were seen at this dose and showed to be reversible during the post weaning period (Day Parturition 35). Marked maternal effects (body weight and liver) have been observed in repeated dose and reproductive toxicity studies. At this 500 mg/kg bw dose Lyral was also detected in the mother's milk. At 25 mg/kg bw a NOAEL for maternal effects has been established and at this dose also no effects on pups are seen. The developmental effects including skin sloughing are much related to maternal toxicity and have shown to be reversible. Therefore Lyral does not have to be classified for developmental toxicity or effects via lactation in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.