Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine and hexane-1,6-diamine and 1,3-phenylenedimethanamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2013-07-25 to 2013-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

: see chapter 'any other information on materials and methods'

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: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: the males were approximately 10 weeks old and the females were approximately 9 weeks old .
- Mean body weight at study initiation: 354 g (range: 330 g to 384 g) for the males and 221 g (range: 183 g to 242 g) for the females.
- Housing: The animals were individually housed, except during pairing and lactation, in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C, except once before treatment (up to 27.3°C)
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 06 August 2013 to 29 September 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. It was ground to a fine powder using a mortar and pestle, and then mixed with the required quantity of vehicle to obtain the high-dose formulation. After 1 hour of magnetic stirring at room temperature, a high-dose formulation sample was used to obtain the low- and intermediate-dose formulations by dilution in the vehicle.
No correction factor was applied.
The test item dose formulations were prepared daily and delivered to the study room at room temperature and protected from light after at least 30 minutes of magnetic stirring at room temperature.

VEHICLE
- Justification for use and choice of vehicle: homogeneous suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. : batch No. MKBH4894V

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred as day 0 post-coitum
- After successful mating each pregnant female was caged individually again

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection analytical method was used.

Homogeneity and stability of the dosage forms: homogeneity of the test substance in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 20 mg/mL and 200 mg/mL. Stability was not assessed because dose formulations were prepared daily.

Test item concentrations: The test item concentrations in the administered dose formulations were analyzed in Weeks 1 and 5 and were within
an acceptable range variations (-4.9% to +3.8%) when compared to the nominal values. No test item was detected in the control dose formulation.

Duration of treatment / exposure:

In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (at least 5 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until day 5 post partum inclusive,
- until sacrifice for female with no delivery.

Day 1 corresponded to the first day of the treatment period.

Frequency of treatment:

7 days/week

Details on study schedule:

- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks

Remarks:

Doses / Concentrations:
0, 100, 300, and 1000 mg/kg bw/day
Basis:
other: nominal doses

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels used in this study were selected on the basis of the results of 2-week preliminary toxicity study performed in the same species and strain. In this study, three groups of five males and five females received the test item by gavage at 100, 300 or 1000 mg/kg/day as a suspension in corn oil. Another group of five males and five females received the vehicle only. There were no mortality and clinical signs in test item-treated groups and no obvious effects on mean body weight and mean food consumption. No test item-related changes were observed in the mean organ weights or at the macroscopic post mortem examination.
Based on these observations, the same dose-levels were chosen for the reproduction/developmental toxicity screening test.

Positive control:

None

Parental animals: Observations and examinations:

AGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day during the treatment period.

BODY WEIGHT: Yes
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the mating period. The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of the mating period, during pregnancy for the period of Days 0-7, 7-14 and 14-20 post coitum and during lactation for the period of Days 1-5 post partum.During the mating period, food consumption was not measured for males or females. Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

WATER CONSUMPTION: No

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pre-mating and mating periods, until the females were mated.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- The following tests were performed on sperm:
=>Sperm motility: At least 200 sperm were assessed for each male. The percentages of motile and non- motile spermatozoa were reported for all groups.

=>Sperm morphology: At least 100 sperm were assessed for each male of control and high-dose groups. Results were expressed as the percentage of spermatozoa in each of the following categories: normal, normally shaped head separated from flagellum, abnormal head separated from flagellum, abnormal head with normal flagellum, abnormal head with abnormal flagellum or abnormal flagellum with normal head.

=>Epididymal sperm count: After thawing, the left cauda epididymis of each male of control and high-dose groups was weighed, minced and homogenized using a Polytron in a saline-triton solution. An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber. Results were expressed as the number of spermatozoa per cauda and per gram of cauda.

=>Testicular sperm count:After thawing, the left testis of each male of control and high-dose groups was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber. Results were expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated.

- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages in control and high-dose groups.

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

Postmortem examinations (parental animals):

SACRIFICE
The males were sacrificed during week 5 of treatment. The body and selected organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on epididymides (only one examined, the other reserved for seminology) and testes (only one examined, the other reserved for seminology) from the males in the control- and high dose groups and on all macroscopic lesions.

The dams were sacrificed on day 6 post-partum, the body and selected organs were weighed and a complete macroscopic examination was performed.
A microscopic examination was performed on ovaries in the control and high-dose groups and on all macroscopic lesions.

GROSS NECROPSY
On completion of the treatment period all surviving F0 males and females were killed by cby an intraperitoneal injection of sodium pentobarbital.
- males: after the end of the pairing period (during week 5 of treatment ),
- females: on day 6 post-partum.,
- females which had not delivered: on day 26 post-coitum, body weight was recorded to check a possible un-noticed delivery.

For females, the numbers of implantation sites in each uterine horn was counted. For females failing to produce a viable litter, the number of uterine implantation sites was re-checked after staining with ammonium sulphide (modification of the Salewski staining technique).

HISTOPATHOLOGY
A microscopic examination was performed on:
- Epididymides, testes and ovaries in animals of the control- and high-dose groups sacrificed at the end of the treatment period,
- all macroscopic lesions of all the animals sacrificed on completion of the treatment period,

ORGAN WEIGHTS:
The body weight of each animal sacrificed as scheduled was recorded before sacrifice, and liver, kidney,epididymides (L and R), prostate, testes (L and R), seminal vesicles and ovaries (L and R) were weighed (wet) as soon as possible after dissection.The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Postmortem examinations (offspring):

SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Statistics:

The heterogeneity of variance between groups were checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Dunnett test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normally distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons was performed using Dunn test.

Reproductive indices:

Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea

Offspring viability indices:

Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 post partum = 100 * (Number of surviving pups on day 4 post partum / Number of live born pups)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths during the study.
There were no clinical signs ascribed to the test item treatment, except ptyalism from 300 mg/kg bw/day which was recorded occasionally in a few animals mostly in the second and third weeks of treatment. This was considered to be of minor toxicological relevance.
Incidental clinical signs noted in test item-treated animals were aggressive behavior, reflux at dosing and cutaneous lesions. The incidences were low and/or not dose-related.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) (See 7.8.1/2 in chapter 'any other information on results incl. tables')
In males and when compared with controls, there were decreases in mean body weight gain over the whole treatment period in test item groups, reaching statistical significance at 1000 mg/kg/day (-15%, - 15% and - 32.3% at 100, 300 and 1000 mg/kg bw/day respectively). This was not considered to be adverse as mean body weight at 1000 mg/kg/day only was considered to be slightly impacted (-9.1% vs. controls, p<0.05) .
There were no effects on mean body weight in females. The slightly higher mean body weight gain (+13% from control data, p<0.05) at 1000 mg/kg bw/day during the gestation period did not have any toxicologically significant impact on the mean body weight.

There were no effects on mean food consumption.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) (See 7.8.1/3 in chapter 'any other information on results incl. tables')
When compared to controls, a few more females (5 vs. 2 in controls) had a prolonged metestrous/diestrous stage for several days at 1000 mg/kg bw/day, explaining the non-statistically significant higher mean estrous cycle length and lower mean number of cycles at this dose-level. As these findings had no impact on the mean reproductive data and were of slight severity, they were considered to be of limited toxicological relevance.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) (See 7.8.1/4 in chapter 'any other information on results incl. tables')
There were no test item treatment-related effects on epididymal sperm motility, morphology and count.
There were no test item treatment-related effects on testicular sperm head count and daily sperm production rate.
When compared with controls, mean testicular sperm count and daily sperm production rate were lower than controls at 1000 mg/kg/day (-18.6% and -19.0%, respectively). However, these parameters can be highly variable in Sprague-Dawley rats and there were no effects on epididymides and testes at microscopic examination. Therefore, the variations noted in mean testicular sperm counts were not considered to be toxicologically significant.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) (See 7.8.1/5 and 7.8.1/6 in chapter 'any other information on results incl. tables')
There were no effects on mating and fertility data.
One female treated at 1000 mg/kg/day was non pregnant (with one corpora lutea only). This was considered to be fortuitous in view of the limited incidence.
There were no effects on mean delivery data.


ORGAN WEIGHTS (PARENTAL ANIMALS)
When compared with controls, there were no test item treatment-related effects on mean organ weights.

Males treated with 1000 mg/kg/day had a minimal statistically significant decrease in body weight ( 9%, p<0.05). This was considered to have contributed to the minimally lower liver absolute weights and higher relative-to-body testes weights in this group. These differences were thus considered not to be related to test item administration, in view also of the absence of gross and/or microscopic correlates in these organs.

The other organ weight changes were not considered to be related to the test item as they were small in amplitude, were not dose-related and/or had no gross or microscopic correlates.


GROSS PATHOLOGY (PARENTAL ANIMALS)
When compared with controls, there were no test item treatment-related macroscopic post-mortem findings.


HISTOPATHOLOGY (PARENTAL ANIMALS)
When compared with controls, there were no test item-related microscopic findings.
The slight vaginal mucification seen in one female given test item at 1000 mg/kg/day was considered to be fortuitous because this finding is not uncommon in the female rats of this strain and age and was not associated with lesions in the ovaries of this female.

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

MORTALITY/VIABILITY/CLINICAL SIGNS (OFFSPRING):
There were no effects on pup viability and no test item treatment-related clinical signs by day 5 post partum.
One control pup was prematurely sacrificed following the absence of anus noted at clinical observation; however, this was not confirmed at necropsy.

BODY WEIGHT (GAIN) (OFFSPRING):
There were no effects on pup mean body weight and mean body weight gain.

GROSS PATHOLOGY (OFFSPRING):
There were no test item-related macroscopic findings at pup necropsy.

SEX RATIO (OFFSPRING):
There were no effects on the percentage of male pups at birth.

Dose descriptor:

NOEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

7.8.1 /2 Parental animals : Body weights and mean body weight changes (g)

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pre-mating (females) or whole study (males)
Day 1	353	358 (+1)	354 (0)	352 (0)	223	221	218	222
Day 15	423	418 (-1)	412 (-3)	396 (-6)	249	253	247	254
Day 36	486	471 (-3)	467 (-4)	442* (-9)	/	/	/	/
Days 1 - 15	+70	+60	+58	+44#	+26	+32	+29	+32
Days 15 - 36	+63	+53	+54	+46	/	/	/	/
Days 1 - 36	+133	+113	+113	+90#	/	/	/	/
Gestation
Day 0 p.c.	/	/	/	/	253	255	250	256
Day 20 p.c.	/	/	/	/	394	410	396	416
Days 0 - 20 p.c.	/	/	/	/	+141	+155	+146	+160*
Lactation
Day 1 p.p.	/	/	/	/	298	309	298	307
Day 5 p.p.	/	/	/	/	309	327	318	324
Days 1 - 5 p.p.	/	/	/	/	+11	+19	+20	+17

p.c.: post-coitum, p.p.: post-partum; /: not applicable.

( ): in brackets percentage of change vs.controls.

*: p<0.05, #: p<0.001.

7.8.1/ 3 Parental animals: Mean estrous cycle data recorded during the premating period

Dose-level (mg/kg/day)	0	100	300	1000
Number of cycles per female	2.9	2.9	3.1	2.3
Cycle length (days)	4.2	4.2	4.4	5.5
Number of females having a mean average cycle of 4-5 days	6	7	5	5

 

7.8.1 /4 Parental animals: Mean sperm analysis data at the end of the male treatment period

Dose-level (mg/kg/day)	0	100	300	1000
% of motile epididymal sperm	96	87	89	88
% of morphologically normal epididymal sperm	96	np	np	97
Mean number of epididymal sperm (10^6/cauda)	114	np	np	121
Mean number of epididymal sperm (10^6/g cauda)	368	np	np	383
Mean number of testicular sperm heads (10^6/g testis)	129	np	np	105
Daily sperm production rate (10^6/g testis/day)	21	np	np	17

No statistics performed; np: not performed

7.8.1 /5 Parental animals: mating and fertility data

Dose level (mg/kg/day)	0	100	300	1000
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Mating index	100%	100%	100%	100%
Mean number of days taken to mate	3.7	2.8	2.4	2.3
Number of pregnant females	10	10	10	9
Fertility index	100%	100%	100%	90%
Number of females with live born pup	10	10	10	9
Gestation index	100%	100%	100%	100%

M: males, F: females.

7.8.1 /6 Parental animals: delivery data

Dose level (mg/kg/day)	0	100	300	1000
Number of females surviving delivery	10	10	10	9
Mean duration of gestation (days)	21.5	21.5	21.5	21.3
Mean number of corpora lutea per female	16.4	18.2	17.1	17.6
Mean number of implantationsper female	15.7	16.9	15.6	17.1
Mean pre-implantation loss (%)per female	3.8	6.9	8.3	2.3
Mean number of pups delivered per female	14.7	15.9	14.3	16.4
Mean post-implantation loss (%) per female^a	6.9	5.8	8.3	3.8

a: manually calculated, no statistics performed.

Conclusions:

Based on the slightly reduced mean body weight in males and the slight increase in mean estrous cycle length in females at 1000 mg/kg bw/day, the dose-level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity and for reproductive performance (mating and fertility), and the No Observed Effect Level (NOEL) for toxic effects on progeny.

Executive summary:

In a screening study for reproductive / developmental effects performed according to OECD 421 guideline , the objective was to evaluate the potential toxic effects of the test substance following daily oral administration (gavage) to male and female rats from before mating, through mating

and, for females, through gestation until day 5 post-partum (p.p.).

Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days before pairing up to the day before necropsy after a minimum treatment period of five consecutive weeks.  Females were treated daily for a minimum of 15 days before pairing, throughout mating and gestation until Day 5 p.p.. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. A constant dose-volume of 5 mL/kg/day was used. The concentrations of the dose formulations were checked in study weeks 1 and 5.

 During the study, clinical condition, bodyweight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults. Estrous cycle was monitored on all females during the pre-mating and mating periods. Gestation length was assessed and parturition observations were performed. In males, sperm investigations were performed at sacrifice. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.

 

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 5 were within the acceptable range of variation when compared to the nominal values (± 15%).

There were no unscheduled deaths.Test item-related clinical signs were limited to ptyalism in a few animals dosed with 300 mg/kg/day and were considered to be of minor toxicological relevance. There were no effects on food consumption and on mean body weight in females. In males, mean terminal body weight was slightly reduced (-9.1% vs. controls, p<0.05) and associated with a lower mean body weight gain over the whole treatment period (-32.3% vs.controls, p<0.001) at 1000 mg/kg/day. A trend toward lower mean body weight gains was also noted in the low- and mid-dose groups (-15% and -15%, vs. controls, respectively, not statistically significant).

During the pre-mating period and when compared with controls, slight and non-statistically significant higher mean estrous cycle length (5.5 vs. 4.2 days) and lower mean number of cycles (2.3 vs. 2.9) at 1000 mg/kg/day were due to a few females (5 vs. controls) with a prolonged metestrous/diestrous stage for several days. These findings were considered to be of limited toxicologically relevance as they had no impact on the mean reproductive data.

In males, there were no toxicologically significant changes in mean sperm parameters (epididymal motility, morphology and count, testicular sperm count).

In adults, there were no test item treatment-related effects on the weight of the selected organs and no macroscopic and microscopic findings.

There were no effects of treatment on offspring survival, sex ratio and offspring bodyweight gain up to Day 5 of age. There were no macropathological findings in the offspring.

 

Based on the slightly reduced mean body weight in males and the slight increase in mean estrous cycle length in females at 1000 mg/kg/day, the dose-level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity and for reproductive performance (mating and fertility), and the No Observed Effect Level (NOEL) for toxic effects on progeny.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Screening reprotoxicity study complete and sufficient to fulfill the REACh annex VIII requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a screening study for reproductive / developmental effects performed according to OECD 421 guideline and in compliance with Good Laboratory Practices , the objective was to evaluate the potential toxic effects of the test substance following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum (p.p.).

Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days before pairing up to the day before necropsy after a minimum treatment period of five consecutive weeks.  Females were treated daily for a minimum of 15 days before pairing, throughout mating and gestation until Day 5 p.p.. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. A constant dose-volume of 5 mL/kg/day was used. The concentrations of the dose formulations were checked in study weeks 1 and 5.

 During the study, clinical condition, bodyweight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults. Estrous cycle was monitored on all females during the pre-mating and mating periods. Gestation length was assessed and parturition observations were performed. In males, sperm investigations were performed at sacrifice. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.

 

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 5 were within the acceptable range of variation when compared to the nominal values (± 15%).

There were no unscheduled deaths.Test item-related clinical signs were limited to ptyalism in a few animals dosed with 300 mg/kg/day and were considered to be of minor toxicological relevance. There were no effects on food consumption and on mean body weight in females. In males, mean terminal body weight was slightly reduced (-9.1%vs.controls, p<0.05) and associated with a lower mean body weight gain over the whole treatment period (-32.3%vs.controls, p<0.001) at 1000 mg/kg/day. A trend toward lower mean body weight gains was also noted in the low- and mid-dose groups (-15% and -15%,vs.controls, respectively, not statistically significant).

During the pre-mating period and when compared with controls, slight and non-statistically significant higher mean estrous cycle length (5.5vs.4.2 days) and lower mean number of cycles (2.3vs.2.9) at 1000 mg/kg/day were due to a few females (5vs.controls) with a prolonged metestrous/diestrous stage for several days. These findings were considered to be of limited toxicologically relevance as they had no impact on the mean reproductive data.

In males, there were no toxicologically significant changes in mean sperm parameters (epididymal motility, morphology and count, testicular sperm count).

In adults, there were no test item treatment-related effects on the weight of the selected organs and no macroscopic and microscopic findings.

There were no effects of treatment on offspring survival, sex ratio and offspring bodyweight gain up to Day 5 of age. There were no macropathological findings in the offspring.

 

Based on the slightly reduced mean body weight in males and the slight increase in mean estrous cycle length in females at 1000 mg/kg/day, the dose-level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity and for reproductive performance (mating and fertility), and the No Observed Effect Level (NOEL) for toxic effects on progeny (Bentz, 2014).

Short description of key information:
In a study performed according to the OECD 421 guideline, the substance was administered daily by oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 5 post-partum at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOAEL for the substance was 1000 mg/kg/day (the limit dose) for reproductive performance and parental toxicity. No effects were observed on progeny.

Justification for selection of Effect on fertility via oral route:
Study performed according to the OECD guideline 421 and GLP compliant.

Justification for classification or non-classification

No effects on reproductive performance and offspring growth and survival were observed at the limit dose of 1000 mg/kg bw/day in a screening study for reproductive /developmental effects performed in rats.

On the basis of this results and according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, no classification is warranted with respect to reproductive toxicity.