reaction products of 2-Propenoic acid (2 moles) and Neopentylglycol hydroxypivalate (1 mole)

Endpoint summary

Description of key information

A combined Repeated dose toxicity study with the Reproduction/Developmental toxicity study is available on the registered substance. Based on the dose-range finding study, a maximal dose of 600 mg/kg/day was chosen for the main OECD 422 study. No adverse toxicity was observed in parental animal, on reproduction parameters and on foetal development at 600 mg/kg/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2017 - 15 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

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: breeder: Janvier, Le Genest-Saint-Isle, France
- Age: at the beginning of the treatment period, the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Mean body weight: at the beginning of the treatment period, the males had a mean body weight of 425 g (range: 392 g to 449 g) and the females had a mean body weight of 262 g (range: 226 g to 296 g).
- Housing: F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 48 x 26.5 x 21 cm, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing of F0 animals was chosen as group housing could adversely affect gestation and lactation, and to avoid aggressive behavior between males around mating.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the males were acclimated to the study conditions for 7 days before treatment and the females were acclimated to the study conditions for 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 20 June 2017 to 15 August 2017.

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study. The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING FORMULATIONS: solution in the vehicle
- Concentration in vehicle: 20, 60 and 120 mg/mL
- Amount of vehicle: 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: remained within an acceptable variation range (-3.4% to +5.4%) when compared to the nominal values.
Homogeneity: diluted analytical samples based on the marker peak prepared from 1.6 mg/mL and 240 mg/mL dose formulations in corn oil were found to be stable for 6 days when they were stored at room temperature and protected from light.

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period (until 1 week),
- until euthanasia (4 weeks in total) (until study Day 28),

In the females:
- 2 weeks before mating,
- during the mating period (until 1 week),
- during gestation,
- during lactation until Day 13 post-partum inclusive,
- until euthanasia for females which had no delivery.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control:

no (not required)

Details on results:

All the results are in the section 7.8.1 (combined study).

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

Based on the experimental conditions of this study:
. the No Observed Effect Level (NOEL) for parental toxicity was considered to be 600 mg/kg/day in females based on the absence of findings at this dose level; in males, given the non-adverse findings in the kidneys, the No Observed Adverse Effect Level (NOAEL) was considered to be 600 mg/kg/day,

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until Day 13 post-partum (p.p.).

This study provides information:

. on the possible health hazards likely to arise from repeated exposure over a relative limited period of time,

. on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item.

The test item was administered daily by the oral route (gavage) at dose levels of 100, 300 or 600 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to one week) until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13post-partum(p.p.) inclusive. Another group of 10 males and 10 females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group.

The actual test item concentrationsin the dose formulations were determined in Weeks 1 and 6 using a validated GC-FID analytical method.

Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stage was determined each morning from two weeks before mating until the females had mated and on Day 14p.p.before euthanasia.

The animals were paired for mating after two weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4p.p.by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on Day 4p.p.were sacrificed and discarded without further examination.

The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13p.p.The physical development of pups was assessed by measuring the anogenital distance on Day 1p.p.and by counting the number of nipples and areolae in male pups on Day 12p.p.

Hematology and blood biochemistry investigations were performed on at least five males and females in each group at scheduled sacrifice. Thyroid hormones (TSH and T4) were determined in males at sacrifice and in pups sacrificed on Day 13p.p.

Males were sacrificed after completion of the mating period. Dams were sacrificed on Day 14p.p.

A full macroscopicpost-mortemexamination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups.

Pups were sacrificed on Day 13p.p.and submitted to a detailed external examination with a particular attention to the external genital organs.

Results

Actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variation (-3.4% to +5.4%) when compared to the nominal concentrations.

 

F0 animals:

No test item-related deaths occurred in males or females during the study.

Ptyalism was observed with a dose-related incidence at all dose levels of the test item. This sign, commonly noted when a test item is administered by gavage, was not considered as an adverse effect.

Body weight and food consumption were unaffected by the test item treatment.

The estrous cycle was not impacted by the test item treatment.

The mating, fertility and delivery data were unaffected by the test item treatment.

Hematology and blood biochemistry parameters were not impacted by the test item treatment.

Thyroid hormone analyses did not reveal any disturbance in F0 males at sacrifice.

At pathology examination, no effects on organ weights and no test item-related macroscopic or microscopic findings were observed in females. In male rats, an increase in renal tubular hyaline droplets was observed. This finding is specific to male rats and has no relevance for human risk assessment.

 

Pups:

Observations of the pups from birth to Day 13p.p.did not show any effects on mortality, viability, clinical signs, body weight evolution, sex ratio or anogenital distance. The only observation consisted of the presence of areolae in one and five pups at 300 and 600 mg/kg/day, respectively. As this finding was isolated with low incidence and was no longer observed the day after in two pups at 600 mg/kg/day, this was considered to be non-adverse.

Thyroid hormone analyses did not reveal any disturbance in pups on Day 13p.p.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until Day 13 post-partum, at dose levels of 100, 300 or 600 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Effect Level (NOEL) for parental toxicity was considered to be 600 mg/kg/day in females based on the absence of findings at this dose level; in males, given the non-adverse findings in the kidneys, the No Observed Adverse Effect Level (NOAEL) was considered to be 600 mg/kg/day,

. the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 600 mg/kg/day based on the absence of effects on mating or fertility at this dose level,

. the NOAEL for toxic effects on progeny was considered to be 600 mg/kg/day based on the absence of adverse findings on pups at this dose level.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

03 March 2017 - 29 March 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Range-finding study

GLP compliance:

no

Remarks:

not required for a range-finding study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks old on the day of treatment
- Mean body weight at study initiation: males: 408 g (range 393 g to 431 g); females: 248 g (range: 238 g to 261 g)
- Fasting period before study: no
- Housing: the animals were housed in fives from the same sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for 6 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: 9 March 2017 to 29 March 2017

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.
The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
A constant dosage-volume of 5 mL/kg/day was used.
Control animals received the vehicle only.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING FORMULATIONS: Solution in the vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, based on the results of a previous acute toxicity study performed in the same species (OECD 423). In this study, two groups of three Sprague Dawley female rats received the test item at 2000 mg/kg by gavage. No mortality and no clinical signs were observed. The maximal tested dose of 1000 mg/kg bw /day is selected.

Rationale for animal assignment: the animals were allocated to groups (by sex) using a computerized stratification procedure so that the average body weight of each group was similar.

Observations and examinations performed and frequency:

Morbidity and mortality:
Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including week-ends.

Clinical signs:
Each animal was observed once a day, at approximately the same time on the days of treatment, for the recording of clinical signs.

Body weight:
The body weight of each animal was recorded:
- once before the beginning of the treatment period,
- on the first day of treatment,
- at least twice a week until the end of the study.

Food consumption:
The quantity of food consumed by the animals in each cage was measured twice a week during the treatment period.
Food consumption was calculated per animal and per day.

Sacrifice and pathology:

ORGAN WEIGHTS:
See Table Tissue Procedure Table
The body weight of each animal was recorded before sacrifice. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

PRESERVATION OF TISSUES:
For all study animals, the tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes, which were fixed in Modified Davidson's Fixative).

HISTOPATHOLOGY:
A microscopic examination was performed on the stomach with forestomach of all study animals.

Other examinations:

no

Statistics:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Ptyalism was observed in 1/5 females given 100 mg/kg/day, and in all males and 3/5 females given 1000 mg/kg/day.
This sign commonly observed when a test item is administered by gavage was not considered to represent an adverse effect.
Tail bent was observed in one control female.
No other clinical signs were recorded.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight and mean body weight gain were unaffected by the test item treatment in both sexes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption was unaffected by the test item treatment in both sexes.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Table 1.
Slightly higher liver absolute and relative-to-body weights were noted in females treated at 1000 mg/kg/day (+19% and +17%, respectively; p<0.01) when compared to controls. No statistically significant changes in liver weights were observed in males.

The other organ weight differences were considered not to be related to the test item administration in view of their low magnitude and the absence of dose-relationship, as noted with the high relative-to-body spleen weights and the low absolute testes weights in males treated at 100 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2.
There were white discolorations in the forestomach from males and females treated at 1000 mg/kg/day associated with brown discolorations or yellow deposits in some males. These findings were considered to be related to the test item administration.
The white and yellow discolorations correlated with hyperkeratosis, while the brown discolorations correlated with erosion/ulcer at microscopic examination.

Yellow deposit in the stomach was observed in two males at 100 mg/kg/day and one male at 1000 mg/kg/day.
This correlated with microscopic minimal increased mucus deposit or increased mucus cells and was considered to be of no toxicological importance in the absence of dose-relationship.

The other macroscopic findings were not considered to be test item-related: red discoloration of thymus, enlarged or reduced kidney, tail deformation and dilatation of uterus.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 3.
There was a spectrum of microscopic lesions in the mucosa and submucosa of the forestomach characterized by:
- minimal to slight erosion/ulcer in 4/5 males treated at 1000 mg/kg/day,
- moderate hyperkeratosis (ortho- and/or parakeratotic) in all males treated at 1000 mg/kg/day and slight hyperkeratosis in all females treated at 1000 mg/kg/day and at minimal severity in 2/5 males treated at 300 mg/kg/day,
- slight to marked squamous cell hyperplasia in all males and females treated at 1000 mg/kg/day and at minimal severity in a single male treated at 300 mg/kg/day,
- minimal to moderate infiltrate of mixed inflammatory cells in almost males and females treated at 1000 mg/kg/day,
- minimal to slight edema in the submucosa from almost males and females treated at 1000 mg/kg/day,
- slight granulomatous inflammation in the submucosa from one male treated at 1000 mg/kg/day.

Among these lesions, the erosion/ulcer was considered to be an adverse effect, in association with hyperplasia of the squamous cells up to grade 4 (marked) observed in males. The other findings were considered to be non-adverse.

Minimal infiltrate of mixed inflammatory cells and edema in the submucosa were noted in the stomach from one male treated at 1000 mg/kg/day, probably by contiguous infiltration from the forestomach in view of the absence of findings at the surface of the mucosa.

There was a minimal erosion in the stomach from one male treated at 300 mg/kg/day. In view of the focal and very limited distribution of this change and in the absence of a similar change at the high dose level, it was considered to be probably unrelated to the test item administration.
The increased mucus deposit or increased mucus cells in the stomach from two males treated at 100 mg/kg/day and one male treated at 1000 mg/kg/day were considered to be of no toxicological importance.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

other: Maximum Tolerated Dose

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Organ:

liver

stomach

other: forestomach

Table 1:

Test item-related organ weight change (in percentages versus controls)

Sex	Male			Female
Group	2	3	4	2	3	4
Dose level (mg/kg/day)	100	300	1000	100	300	1000
Exam. animals / Num. of animals	5/5	5/5	5/5	5/5	5/5	5/5
- Final body weight	-1	0	-3	0	-1	+2
- Liver
. absolute	+8	+4	+10	+2	+4	+19**
. relative	+9	+4	+13	+2	+5	+17**

Statistically significant from controls: **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Table 2:

Incidence of test item-related gross changes

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Number of animals	5	5	5	5	5	5	5	5
Forestomach
- White discoloration	-	-	-	5	-	-	-	3
- Brown discoloration	-	-	-	2	-	-	-	-
- Yellow deposit	-	-	-	1	-	-	-	-

-: no findings.

Table 3:

Incidence and severity* of main test item-related microscopic changes in the forestomach

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Number of animals	5	5	5	5	5	5	5	5
Erosion/ulcer
grade 1	-	-	-	2	-	-	-	-
grade 2	-	-	-	2	-	-	-	-
Hyperkeratosis
grade 1	-	-	2	-	-	-	-	-
grade 2	-	-	-	-	-	-	-	5
grade 3	-	-	-	5	-	-	-	-
Hyperplasia; squamous cell
grade 1	-	-	1	-	-	-	-	-
grade 2	-	-	-	-	-	-	-	4
grade 3	-	-	-	4	-	-	-	1
grade 4	-	-	-	1	-	-	-	-
Infiltrate; mixed cells
grade 1	-	-	-	-	-	-	-	2
grade 2	-	-	-	4	-	-	-	2
grade 3	-	-	-	1	-	-	-	-
Edema; submucosa
grade 1	-	-	-	1	-	-	-	2
grade 2	-	-	-	4	-	-	-	2

*: scale of grades used in this study: 1 (minimal); 2 (slight); 3 (moderate); 4 (marked).

-: not observed.

Conclusions:

The potential toxicity of the test item, was evaluated after daily administration (gavage) to Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks.
At 1000 mg/kg/day, the test item was clinically well tolerated. Slightly higher liver weights were noted in females, and white discolorations were noted in the forestomach from both sexes associated with brown discolorations or yellow deposits in two or one males, respectively. At microscopic examination, there was adverse erosion/ulcer in 4/5 males treated at 1000 mg/kg/day, associated mainly with hyperkeratosis, squamous cell hyperplasia, infiltrate of mixed inflammatory cells and edema in the submucosa in males and females.
At 100 and 300 mg/kg/day, the test item was clinically well tolerated and did not induce test item-related macroscopic post mortem changes. At microscopic examination, there was non-adverse minimal hyperkeratosis and squamous cell hyperplasia in occasional males treated at 300 mg/kg/day.
Based on the results of this study, the dose level of 1000 mg/kg/day is considered to be higher than the Maximal Tolerated Dose (MTD).

Executive summary:

The objective of this preliminary study was to evaluate the potential toxicity of the test item, following daily oral administration (gavage) to rats for 2 weeks in order to assist the selection of dose levels for a further OECD 422 study to be performed in this species.

 

Methods

Three groups of five male and five female Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks.

Another group of five males and five females received the vehicle alone (corn oil) under the same experimental conditions and acted as a control group. The test item was administered as a solution in the vehicle under a constant dosage-volume of 5 mL/kg/day.

The animals were observed daily for mortality and clinical signs. Body weight was recorded once pre-test, on the first day of treatment and then twice a week until the end of the study. Food consumption was recorded twice aweek during the treatment period.

On completion of the treatment period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. The heart, kidneys, liver, ovaries, spleen, stomach and testes were weighed and selected tissue specimens were preserved. A microscopic examination was performed on the stomach with forestomach of all study animals.

Results

No unscheduled deaths occurred during the study.

Ptyalism was observed in 1/5 females given 100 mg/kg/day and in all males and 3/5 females given 1000 mg/kg/day. This sign commonly observed when a test item is administered by gavage was not considered to represent an adverse effect.

Body weight and food consumption were unaffected by the test item treatment.

Slightly higher liver weights were noted in females treated at 1000 mg/kg/day.

Test item-related white discolorations were noted in the forestomach from males and females treated at 1000 mg/kg/day associated with brown discolorations and/or yellow deposits in some males. At microscopic examination, there was adverse erosion/ulcer in 4/5 males treated at 1000 mg/kg/day, associated mainly with hyperkeratosis, squamous cell hyperplasia, infiltrate of mixed inflammatory cells and edema in the submucosa in males and females treated at 1000 mg/kg/day. At 300 mg/kg/day, the test item induced non-adverse minimal hyperkeratosis and squamous cell hyperplasia in occasional males. At 100 mg/kg/day, there were no test item-related microscopic changes.

Conclusion

The potential toxicity of the test item was evaluated after daily administration (gavage) to Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/dayfor 2 weeks.

At 1000 mg/kg/day, the test item was clinically well tolerated. Slightly higher liver weights were noted in females, and white discolorations were noted in the forestomach from both sexes associated with brown discolorations or yellow deposits in two or one males, respectively. At microscopic examination, there was adverse erosion/ulcer in 4/5 males treated at 1000 mg/kg/day, associated mainly with hyperkeratosis, squamous cell hyperplasia, infiltrate of mixed inflammatory cells and edema in the submucosa in males and females.

At 100 and 300 mg/kg/day, the test item was clinically well tolerated and did not induce test item-related macroscopic post-mortem changes. At microscopic examination, there was non-adverse minimal hyperkeratosis and squamous cell hyperplasia in occasional males treated at 300 mg/kg/day.

Based on the results of this study, the dose level of 1000 mg/kg/day is considered to be higher than the Maximal Tolerated Dose (MTD).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is considered to be reliable with a klimish score of 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

14 -day repeated toxicity study (Michel 2017) = dose range-finding study for OECD 422 study

The potential toxicity of the test item, was evaluated after daily administration (gavage) to Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks.

At 1000 mg/kg/day, the test item was clinically well tolerated. Slightly higher liver weights were noted in females, and white discolorations were noted in the forestomach from both sexes associated with brown discolorations or yellow deposits in two or one males, respectively. At microscopic examination, there was adverse erosion/ulcer in 4/5 males treated at 1000 mg/kg/day, associated mainly with hyperkeratosis, squamous cell hyperplasia, infiltrate of mixed inflammatory cells and edema in the submucosa in males and females.

At 100 and 300 mg/kg/day, the test item was clinically well tolerated and did not induce test item-related macroscopic post mortem changes. At microscopic examination, there was non-adverse minimal hyperkeratosis and squamous cell hyperplasia in occasional males treated at 300 mg/kg/day.

Based on the results of this study, the dose level of 1000 mg/kg/day is considered to be higher than the Maximal Tolerated Dose (MTD).

A combined Repeated dose toxicity study with the Reproduction/Developmental toxicity study (Haag 2018) = OECD 422

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until Day 13 post-partum (p.p.).

Three groups of ten male and ten female Sprague-Dawley rats received the test item. The test item was administered daily by the oral route (gavage) at dose levels of 100, 300 or 600 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to one week) until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13post-partum(p.p.) inclusive. Another group of 10 males and 10 females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group.

The actual test item concentrationsin the dose formulations were determined in Weeks 1 and 6 using a validated GC-FID analytical method.

Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stage was determined each morning from two weeks before mating until the females had mated and on Day 14p.p.before euthanasia.

The animals were paired for mating after two weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p.by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on Day 4p.p.were sacrificed and discarded without further examination.

The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13 p.p.The physical development of pups was assessed by measuring the anogenital distance on Day 1p.p.and by counting the number of nipples and areolae in male pups on Day 12 p.p.

Hematology and blood biochemistry investigations were performed on at least five males and females in each group at scheduled sacrifice. Thyroid hormones (TSH and T4) were determined in males at sacrifice and in pups sacrificed on Day 13 p.p.

Males were sacrificed after completion of the mating period. Dams were sacrificed on Day 14 p.p.

A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups.

Pups were sacrificed on Day 13 p.p.and submitted to a detailed external examination with a particular attention to the external genital organs.

No test item-related deaths occurred in F0 males or females during the study. Ptyalism was observed with a dose-related incidence at all dose levels of the test item. This sign, commonly noted when a test item is administered by gavage, was not considered as an adverse effect. Body weight and food consumption were unaffected by the test item treatment. The estrous cycle was not impacted by the test item treatment. The mating, fertility and delivery data were unaffected by the test item treatment.

Hematology and blood biochemistry parameters were not impacted by the test item treatment. Thyroid hormone analyses did not reveal any disturbance in F0 males at sacrifice. At pathology examination, no effects on organ weights and no test item-related macroscopic or microscopic findings were observed in females. In male rats, an increase in renal tubular hyaline droplets was observed. This finding is specific to male rats and has no relevance for human risk assessment.

 

Observations of the pups from birth to Day 13 p.p.did not show any effects on mortality, viability, clinical signs, body weight evolution, sex ratio or anogenital distance. The only observation consisted of the presence of areolae in one and five pups at 300 and 600 mg/kg/day, respectively. As this finding was isolated with low incidence and was no longer observed the day after in two pups at 600 mg/kg/day, this was considered to be non-adverse.

Thyroid hormone analyses did not reveal any disturbance in pups on Day 13 p.p.

 

Based on the experimental conditions of this study:

. the No Observed Effect Level (NOEL) for parental toxicity was considered to be 600 mg/kg/day in females based on the absence of findings at this dose level; in males, given the non-adverse findings in the kidneys, the No Observed Adverse Effect Level (NOAEL) was considered to be 600 mg/kg/day,

. the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 600 mg/kg/day based on the absence of effects on mating or fertility at this dose level,

. the NOAEL for toxic effects on progeny was considered to be 600 mg/kg/day based on the absence of adverse findings on pups at this dose level.

Justification for classification or non-classification

Based on the available data, no classification for repeated toxicity is required for the registered substance according to the Regulation EC N°1272/2008.