Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
One-generation study: NOAEL parental toxicity, reproduction and offspring development = 1000 mg/kg bw/day (OECD 415, GLP, K, rel.1) 
Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2001-11-23 to 2002-03-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 415 and in compliance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Version / remarks:
1983
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on: 2000-02-28 / signed on 2000-04-26)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Remarks:
Crl: CD (SD) IGS BR
Details on species / strain selection:
The rat was selected as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Crl: CD (SD) IGS BR strain
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent- UK
- Age at study initiation: no data
- Weight at study initiation: Males: 185 to 259 g; Females: 187 to 277g
- Fasting period before study: none
- Housing: Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis. Following evidence of successful mating, the males were returned to their original cages and transferred to a separate animal room of comparable conditions. The females were housed, individually, in polypropylene cages with solid floors and stainless steel tops. Mated females were given softwood chips, as bedding throughout gestation and lactation.
- Diet (e.g. ad libitum): ad libitum (pelleted Rat and Mouse VRF1C Diet (Charles River UK Limited, Margate, Kent))
- Water (e.g. ad libitum): ad libitum
- Acclimation period: seventeen days for males and thirty one days for females


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
(On isolated occasions the room temperature and/or humidity fell outside the protocol target limits but this was considered not to have affected the purpose or integrity of the study)
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 2001-11-06 To: 2002-03-30
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentration as a solution in 0.5 % carboxymethyl cellulose (vehicle). For each dose level a separate aliquot of test material was weighed into the appropriate container. The vehicle was added and mixed using a Silverson homogeniser to ensure a homogeneous suspension was formed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not soluble in water
- Concentration in vehicle: 0, 5, 25 and 100 mg/L
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1 (1 female + 1 male per cage)
- Length of cohabitation: until evidence of successful mating (up to three weeks)
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating
- After successful mating each pregnant female was caged (how): Following evidence of successful mating, mated females were separated from male and housed individually during the period of gestation and lactation. The males were returned to their original cages and transferred to a separate animal room of comparable conditions.
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation were taken once per week during the first four weeks and approximately once every month until study completion and analysed by gas chromatography (GC) using an external standard technique. The results indicate that the prepared formulations were within acceptable limits of the nominal concentration. The analytical method used has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
(P) Females: 17 days prior to pairing, then throughout mating, gestation and lactation.
(P) Males: 73 days before mating, then like females
Frequency of treatment:
Once daily
Details on study schedule:
Male animals were dosed for 73 days and female animals were dosed for 17 days, at their appointed dose levels, prior to pairing.
Parental males and females were paired within their respective dose groups for up to twenty one days.
Following evidence of mating, the animals were separated and males returned to their holding cages.
The pregnant females were allowed to deliver their offspring. The offspring were observed for growth and development during lactation.
At weaning on Day 21 (or as near to this date as possible) post partum the surviving offspring were killed and examined macroscopically post mortem.
The surviving adult Parental animals were killed and examined macroscopically post mortem.
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
28 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations tested in this study were determined from a previous 28-d toxicity study (OECD 407 Guideline) performed with the same test material on the same rat strain and for which a NOEL of 250 mg/kg bw was determined.
- Rationale for animal assignment (if not random): random
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the normal week and once daily during the week-end and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, immediately before, immediately after and one hour after dosing for clinical signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during maturation and mating period. Following mating the parental males were weighed weekly until termination. Parental generation females showing evidence of mating were weighed on days 1, 4, 7, 14 and 21 post coitum. Parental generation females with a live litter were weighed on Days 1, 4, 7, 14 and 21 post partum.

FOOD CONSUMPTION:
During the maturation periods food consumption was recorded for each cage of parental generation adults. For parental generation females showing evidence of mating, food consumption was recorded for the periods covering days 1 to 7, 7 to 14 and 14 to 21 post coitum. For parental generation females with live litters, food consumption was recorded for the periods covering Days 1 to 7 and 7 to 14 postpartum.
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded.
Sperm parameters (parental animals):
Not Applicable/Not required in OECD 415 Guideline
Parameters examined in male parental animals: testis weight, epididymis weight, seminal vesicles (with coagulating gland) weight, spermatozal content
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- Number (daily)
- Sex (PND 1 and 21)
- Bodyweight (PND 1, 4, 7, 14 and 21)
- Clinical signs (Daily)
- Physical development including detachment of pinna, tooth eruption, eye opening
- Reflexological assessment including surface righting reflex (PND 1), mid-air righting reflex (PND 17), startle reflex (PND 21) and pupil reflex (PND21).

GROSS EXAMINATION OF DEAD PUPS:
yes, all offspring that died, or were killed in extremis during the lactation period were examined macroscopically and externally.
Postmortem examinations (parental animals):
All adult animals, killed in extremis or found dead during the course of the study, were examined macroscopically for internal and external abnormalities. All significant abnormalities were retained in fixative for possible further study.

SACRIFICE
Following successful weaning of offspring, all the surviving adults, including non-fertile animals, were killed by carbon dioxide asphyxiation, followed by cervical dislocation.

GROSS NECROPSY
All animals were examined macroscopically for both internal and external abnormalities. Selected organs and tissues were retained in fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights were determined for: Epididymides, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles (with coagulating gland), Testes, Uterus with cervix.
Histopathology was performed on: Cervix, Coagulation gland, Epididymides, Kidneys, Liver, Pituitary gland, Prostate, Seminal vesicles, Significant abnormalities, Testes, Uterus, Vagina, Ovaries. Initially the tissues from high dose and control animals were processed and embedded in paraffin wax BP (mp 56 °C). Sections of the tissues were taken at 5 µm thickness, mounted on glass slides and stained with haematoxylin and eosin. In addition, following the results of the initial examination, the target organs from the low and intermediate dose groups were similarly processed.

The sections of reproductive and target organs from control and high dose animals were examined microscopically by a pathologist. Examination of the target organs was extended to the low and intermediate dose groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem macroscopic examinations for internal and external abnormalities.
Statistics:
The following parameters were analysed statistically, where appropriate using the test methods outlined as follows:
Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight, offspring landmarks of physical development.
Adult precoital intervals, female gestation lengths, offspring reflexological responses and litter sex ratios: Individual values were compared using the Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, multiple comparisons of control values against treated group values was performed using Mann-Whitney "U" test.
Histopathology: Chi-squared analysis 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 for the comparison of severity grades for the more frequently observed conditions.
Reproductive indices:
For each group the following were calculated.
Pre-coital interval: calculated as the time elapsing between initial pairing and the observation of positive mating
Mating Index % = number of animals mated/number of animals paired * 100
Pregnancy index % = Number of pregnant females / Number of animals mated * 100
Gestation length: calculated as the number of days of gestation including the days for observation of mating at the start of parturition. When the start of parturition occurred overnight, the total was adjusted by substracting half a day.
Parturition index % = Number of females delivering live pups / Number of pregnant females * 100
Offspring viability indices:
The following indices were calculated for each group from individual data.
Live birth index = Number of pups alive on Day 1 / Number of pups born * 100
Viability Index 1 (%) = Number of pups alive on Day 4/Number of pups alive on Day 1 * 100
Viability Index 2 (%) = Number of pups alive on Day 7/Number of pups alive on Day 4 * 100
Viability Index 3 (%) = Number of pups alive on Day 14/Number of pups alive on Day 7 * 100
Viability Index 4 (%) = Number of pups alive on Day 21/Number of pups alive on Day 14 * 100
Viability Index 5 (%) = Number of viable pups at weaning/Number of pups alive on Day 1 * 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d there was evidence of increased salivation, predominantly post dosing for males and females. For males only, there was increased salivation pre-dosing which was at a lower incidence and frequency compared to post-dosing salivation. All other clinical findings were those commonly observed on this type of study.
At 250 mg/kg bw/d the majority of males showed evidence of increased salivation immediately post dosing. The incidence and frequency were lower than was seen at the highest dose level. Only one female showed similar findings. There were no other significant clinical findings.
At 50 mg/kg bw/d one male only showed evidence of increased salivation immediately post dosing. There were no other significant clinical findings for this group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 1000 and 250 mg/kg bw/d there were no mortalities during the course of the study.
At 50 mg/kg bw/d one female was found dead during the gestation phase of the study. There was no previous clinical history. Post mortem macroscopic findings showed significant changes within the gastro-intestinal tract including gaseous distension of the small intestine and discolouration of the contents of the intestines.
At 0 mg/kg bw/d there were no mortalities.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect
Food consumption and compound intake (if feeding study):
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 7.8.1/1
At 1000 mg/kg bw/d there was an increase in liver and kidney weight for males only compared to control values. The differences were statistically significant (p<0.01 and p<0.001 respectively) for absolute values and also significant (p<0.001) for organ weights as a percentage of bodyweight. A statistically significant difference in female absolute pituitary weight (p<0.05) compared to control values was concluded to be incidental as no effect on relative organ weight value, no dosage related trend and no effect on male pituitary weight were observed.
At 250 mg/kg bw/d there was an increase in male kidney weight compared to control values. The difference was statistically significant (p<0.01) for absolute kidney weight but not significant when compared as a percentage of bodyweight. A slight increase in male liver weight was of note although the difference, compared to control values was not statistically significant. A slight increase in male absolute epididymis weight which was statistically significant (p<0.05) was considered to be fortuitous as no dosage related trend was observed. There were no significant differences in female organ weight values compared to controls.
At 50 mg/kg bw/d there were no significant differences in male or female organ weight values compared to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no significant treatment-related macroscopic findings for males or females. The findings observed were those commonly encountered in this type of study.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See table 7.8.1/2
At 1000 mg/kg bw/d there were no treatment-related histopathological changes associated with reproductive organs. Treatment-related changes were present in the kidney of males and liver of both sexes. A statistically significant increase in the incidence and severity of globular accumulations of eosinophilic material in the renal tubular epithelium (p<0.001) and a greater severity and prevalence of groups of basophilic renal tubules (p<0.001) were observed when compared to control values. Generalised hepatocyte enlargement was observed for males and females at a statistically significant incidence (p<0.01 and p<0.001 respectively). In addition, associated low severity grades of glycogen type hepatocyte vacuolation were observed for males and females (p<0.01 and p<0.05 respectively).
At 250 mg/kg bw/d, a statistically significant incidence and severity of globular accumulations of eosinophilic material was observed in the renal tubular epithelium of males only (p<0.001) together with higher severity grades and prevalence of groups of basophilic tubules. Generalised heptatocyte enlargement was observed for males only at an incidence that was statistically significant (p<0.05) with low severities of glycogen type hepatocyte vacuolation (p<0.01).
At 50 mg/kg bw/d a statistically significant increase in incidence and severity of globular accumulations of eosinophilic material was observed in the kidneys for males only (p<0.001).
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no significant treatment-related effects upon mating performance or fertility. The intergroup distribution of precoital intervals showed that the majority of pairings resulted in positive evidence of mating within four days after pairing.
There were no significant treatment-related effects upon pregnancy or parturition.
At 50 mg/kg bw/d one female was found dead during parturition but this was not attributed to a test material effect upon pregnancy.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no significant treatment-related effects upon offspring clinical condition throughout lactation; as indicated by comparable incidence and severity ofclinical findings observed.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no significant treatment-related effects upon litter size at birth and subsequent offspring viability during lactation.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant treatment-related effects upon individual offspring weight gain during lactation.
The onset of tooth eruption of group 3 offspring (250 mg/kg bw/d) was significantly different (p<0.05) from the control: 10.6 days compared to 11.4 days. However at completion this difference was no longer significant: 14.4 compared to 14.8 days. This isolated statistically significant differences in onset of landmarks of development was considered to be incidental events that were unrelated to treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no significant. treatment-related .differences in the type and incidence of macroscopic post mortem findings for offspring that died during the course of lactation or for offspring at terminal necropsy.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no significant treatment-related effects upon offspring reflexological responses.
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 7.8.1/1: Kidneys and Liver weight of parental males

Dose level (mg/kg bw/d)

Kidneys

Liver

Weight (g)

% of bodyweight

Weight (g)

% of bodyweight

Males

0

Mean

SD

N

3.872

0.5117

28

0.637

0.0624

28

20.414

0.3163

28

3.337

0.3161

28

50

Mean

SD

N

4.031

0.2891

28

0.667

0.0563

28

20.802

2.3768

28

3.426

0.2510

28

250

Mean

SD

N

4.227**

0.5127

28

0.674

0.0610

28

22.213

3.3218

28

3.518

0.2809

28

1000

Mean

SD

N

4.389***

0.3850

28

0.737***

0.0466

28

23.271**

2.4034

28

3.901***

0.2629

28

*= significantly different from control value (p<0.05)

** = significantly different from control value (p<0.01)

*** = significantly different from control value (p<0.001)

Table 7.8.1/2 : Histopathological findings in parental animals

Histopathological Finding

Dose Level (mg/kg bw/d)

Males (28 animals/dose)

Females (28 animals/dose)

0

50

250

1000

0

50

250

1000

Kidney

Groups of basophilic tubules

 

 

+

+++

 

 

 

 

-         Absent

13

13

8

3

26

26

25

28

-         Minimal

15

13

14

13

2

2

3

0

-         Slight

0

1

4

12

0

0

0

0

-         Moderate

0

1

2

0

0

0

0

0

Globular accumulations of eosinophilic

material

 

+++

+++

+++

 

 

 

 

-         Absent

17

3

2

2

 

 

 

 

-         Minimal

6

18

18

10

 

 

 

 

-         Slight

5

5

8

12

 

 

 

 

-         Moderate

0

2

0

4

 

 

 

 

 

Liver

Hepatocyte vacuolation

 

 

--

--

 

 

 

-

-         Absent

2

2

3

13

9

9

9

18

-         Minimal

15

16

23

10

18

18

17

10

-         Slight

11

10

2

5

1

1

2

0

Hepatocyte enlargement

 

 

+

++

 

 

 

+++

-         Absent

28

26

22

19

25

20

22

12

-         Minimal

0

2

6

8

3

7

3

13

-         Slight

0

0

0

1

0

1

3

3

 +/- = significantly greater/lower from control value (p<0.05)

++/-- = significantly greater/lower from control value (p<0.01)

+++/--- = significantly greater/lower from control value (p<0.001)

Conclusions:
Based on the results of this study, the No Effect Level (NOAEL) for parental toxicity, reproduction and offspring development is therefore 1000 mg/kg bw/d. Under the test conditions, the test material is not classified according to the Regulation (EC) No. 1272/2008 (CLP) and to to the GHS.
Executive summary:

In a one-generation reproduction toxicity study performed in accordance with OECD test guideline No. 415 and in compliance with GLP, the test material diluted in carboxymethylcellulose (CMC) was administered orally, by gavage, to groups of twenty-eight Sprague-Dawley Crl: CD (SD) IGS BR rats per sex throughout maturation, mating, gestation and lactation. The dose levels were 50, 250 and 1000 mg/kg bw/day with a similar sized control group receiving vehicle alone. Following at least ten and two weeks of dosing respectively, male and female rats were paired within their dose groups to produce litters. At weaning of the offspring, all surviving animals were killed and examined macroscopically.

Parental animals were observed daily for clinical signs. Bodyweights and food consumption were recorded weekly during the maturation phase which was continued for males after the mating phase. Mated females were weighed and food consumption recorded on specific days post coitum and post partum. The offspring were observed daily for clinical signs. The litter size and individual pup bodyweights were recorded on specific days post partum. During the lactation period, the offspring were observed for intra-litter onset and duration of landmarks or physical development. On specific days of lactation, reflexological assessment of offspring was performed.  

Post mortem macroscopic examinations were performed on all adults and offspring, including decedents. Reproductive and potential target organs were weighed from all parental animals and were then preserved in fixative. Histopathology was carried out on reproductive and target organs from control and high dose groups parental animals. The target organs were examined from the low and intermediate groups.

 

For all dose levels, particularly 250 mg/kg bw/day and above there was evidence of increased salivation, predominantly post dosing. The incidence and frequency were dosage-related. The finding was considered to be an adaptation to administration of an unpleasant-tasting material. There were no other significant effects upon adults during the in-life phase of the study.

There were no treatment-related effects upon reproductive performance or fertility and upon offspring viability, growth or development.

Post mortem findings showed no treatment-related effects upon the reproductive organs.

Significant liver weight increases were observed in males dosed at 1000 mg/kg bw/day. Histopathology showed higher incidence of hepatocyte enlargement associated with lower severity grades of glycogen type hepatocyte vacuolation at 1000 mg/kg bw/day for both males and females, and at 250 mg/kg bw/day for males only. It is assumed that the observed hepatocyte enlargement occurs as an induction of the microsomal drug metabolizing enzyme systems caused by the treatment and is considered as cellular adaptation phenomena in the absence of associated inflammatory or degenerative changes.

Significant kidney weight increases were also observed in males dosed at both highest doses. These increases were accompanied by eosinophilic accumulations together with an increased prevalence of basophilic tubules in the kidney of males only. In the case of the renal tubular accumulations of eosinophilic material (considered by the study author to be alpha-2 microglobin), this change is exclusive to male rats. Tubular basophilia is known to be an early stage of chronic progressive nephrosis which is frequently seen in Sprague-Dawley rat.

Under the test conditions, the No Effect Level (NOAEL) for parental toxicity, reproduction and offspring development is therefore 1000 mg/kg bw/day.

The one-generation reproduction toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a one-generation reproduction toxicity study (OECD 415) in rats.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 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

A key study was identified (Safepharm, 2003, rel.1). In this one-generation reproduction toxicity study performed in accordance with OECD test guideline No. 415 and in compliance with GLP, the substance diluted in carboxymethylcellulose (CMC) was administered orally, by gavage, to groups of twenty-eight Sprague-Dawley Crl: CD (SD) IGS BR rats per sex throughout maturation, mating, gestation and lactation. The dose levels were 50, 250 and 1000 mg/kg bw/day with a similar sized control group receiving vehicle alone. Following at least ten and two weeks of dosing respectively, male and female rats were paired within their dose groups to produce litters. At weaning of the offspring, all surviving animals were killed and examined macroscopically. Histopathology was carried out on reproductive and target organs from control and high dose groups parental animals. The target organs were examined from the low and intermediate groups.  

For all dose levels, particularly 250 mg/kg bw/day and above there was evidence of increased salivation, predominantly post dosing. The incidence and frequency were dosage-related. The finding was considered to be an adaptation to administration of an unpleasant-tasting material.

There were no other significant effects upon adults during the in-life phase of the study. There were no treatment-related effects upon reproductive performance or fertility and upon offspring viability, growth or development. Post mortem findings showed no treatment-related effects upon the reproductive organs.

Significant liver weight increases were observed in males dosed at 1000 mg/kg bw/day. Histopathology showed higher incidence of hepatocyte enlargement associated with lower severity grades of glycogen type hepatocyte vacuolation at 1000 mg/kg bw/day for both males and females, and at 250 mg/kg bw/day for males only. It is assumed that the observed hepatocyte enlargement occurs as an induction of the microsomal drug metabolizing enzyme systems caused by the treatment and is considered as cellular adaptation phenomena in the absence of associated inflammatory or degenerative changes.

Significant kidney weight increases were also observed in males dosed at both highest doses. These increases were accompanied by eosinophilic accumulations together with an increased prevalence of basophilic tubules in the kidney of males only. In the case of the renal tubular accumulations of eosinophilic material (considered by the study author to be alpha-2 microglobin), this change is exclusive to male rats. Tubular basophilia is known to be an early stage of chronic progressive nephrosis which is frequently seen in Sprague-Dawley rat.

Under the test conditions, the No Effect Level (NOAEL) for parental toxicity, reproduction and offspring development is therefore 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity study in rat: NOAEL = 1000 mg/kg bw/day (OECD 414, GLP, K, rel.1)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 29 May to 30 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD TG No. 414 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other:
Version / remarks:
OPPTS 870.3800 Reproduction and Fertility Effects, August 1998.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 210 g to 319 g.
- Fasting period before study: none
- Housing: individually in solid-floor cages with appropriate bedding provided
- Diet: ad libitum (pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England)
- Water: ad libitum
- Acclimation period: at least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 23°C
- Humidity (%): 40% to 70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2018-05-29 To: 2018-08-30
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (high viscosity)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A weighed quantity of test item was added to the final preparation container, then 20 % of the final required volume of vehicle was added and stirred vigorously to form an emulsion. After further addition of vehicle and mixing, the resultant suspension was mixed with a laboratory homogeniser and then stirred for a minimum of 20 minutes thereafter.
Formulations were divided into daily aliquots and were stored refrigerated (2 °C to 8 °C) until at least 20 minutes before dosing on the day of use, when they were stirred before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on previous toxicology studies
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability: Stability of test item formulations prepared at concentrations of 1 to 100 mg/mL, spanning those used in this study (10 to 100 mg/mL), were examined in an earlier formulation validation study. Those formulations were found to be stable for 8 and 14 days when stored at room temperature (15 to 25 °C) and refrigerated (2 to 8 °C), respectively.
- Homogeneity and achieved concentrations: Duplicate samples were taken from the top, middle and bottom of each test item formulation prepared for use on the first day of dosing and on one occasion towards the end of the dosing period. One set of samples was analysed using a validated method (3) to confirm homogeneity and achieved concentrations by analysis by a gas chromatographic assay using flame ionisation detection. On these occasions, duplicate samples were also taken from the vehicle used to dose Controls and were analysed to confirm absence of test item.
Analysis of the samples confirmed homogeneity and achieved concentrations as well as absence of the test item in the vehicle. All remaining samples were retained as a contingency and stored refrigerated (approximately 2 ºC to 8 °C) and discarded once the final formulation analysis results were accepted.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
From Day 6 to Day 19 of gestation inclusive
Frequency of treatment:
Once daily
Duration of test:
From Day 6 to Day 20 of gestation
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:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of results from existing toxicity data
- Rationale for animal assignment (if not random): random
Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and morbidity and were given a detailed clinical examination daily from the start of treatment

BODY WEIGHT: Yes
- Time schedule for examinations: by the supplier on Day 0 of gestation. At Sequani, body weights were recorded for all females daily from Day 5 to Day 20 of gestation.

FOOD CONSUMPTION : Yes
- The amount of food consumed by each animal was recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 of gestation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The cranial and thoracic cavities were opened, a full internal examination was performed, and all macroscopic abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes (right and left horn)
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate.
Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1.
Depending on the nature of the data set that was to be analysed, appropriate tests were applied, Where parametric tests were appropriate they were preceded
by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied.
Proportions of foetuses affected were treated as continuous non-parametric data, using onesided step-wise Jonckheere Tests.
Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.
Indices:
Pre-implantation loss (%) = (no. of corpora lutea – no. of implantation sites) / no. of corpora lutea x 100
Post-implantation loss (%) = (no. of implantation sites – no. of live foetuses) / no. of implantation sites x 100
Historical control data:
Included in the report
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in the surviving animals following administration of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were 2 deaths during the study, both were unrelated to the test item. Female 55, given 300 mg/kg/day, was euthanised on Day 12 of gestation due to 9 % body weight loss between Days 11 and 12 of gestation and clinical signs of pale extremities, cold body surface, rapid breathing and hunched postured. At necropsy, the lungs were red and the thoracic cavity had clear fluid contents.
Female 74, given 1000 mg/kg/day, was euthanised on Day 14 of gestation due to red discharge from the vulva. At necropsy, there was red contents in the vagina and the thymus was large.
Due to their isolated nature, these deaths were considered not to be related to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following the first day of administration, at 1000 mg/kg/day, there was a slight, but statistically significant, mean body weight loss (p≤0.001). In the group given 300 mg/kg/day, mean body weight gain was also slightly lower than Controls (p≤0.05) but from Day 7 of gestation, mean body weight gain in all groups was similar to, or in excess of, Controls such that by Day 20 of gestation, all body weight values were comparable across all groups.
There was no effect on the terminal body weight adjusted for the weight of the gravid uterus.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, group mean food intake was significantly lower than Controls from Day 6 to Day 9 of gestation (p≤0.001). Thereafter food intake was similar to Controls.
There was no effect on food intake following administration at 100 or 300 mg/kg/day
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:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
On Day 20 of gestation, there were 20, 21, 21 or 20 females pregnant with live foetuses in the groups given 0, 100, 300 or 1000 mg/kg/day, respectively. There was no effect of test item
administration on the pregnancy data.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effect observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There was no effect of the test item administration on the overall incidences of minor and variant abnormalities.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant increases in a few minor and variant skeletal findings, largely relating to changes in the extent of ossification (p<0.05 and
p<0.01). These abnormalities were largely within the background data range and/or had no dose response. Only 3 isolated skeletal changes were outside the background range and statistically significantly higher than Controls at 300 or 1000 mg/kg/day (incomplete ossification of the cervical vertebral neural arch, interrupted costal cartilage and incomplete ossification of the sternebra). Although statistically significantly higher than Controls at 300 or 1000 mg/kg/day, these findings are isolated and disparate in nature and are considered unrelated to the test item.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Administration of the test item to the pregnant Crl:CD(SD) rat at 100, 300 or 1000 mg/kg bw/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, was generally well tolerated at all dose levels. A mild and transient effect on maternal food consumption and weight gain was observed over the first day of administration on Day 6 of gestation, with no effect on the developing conceptus or foetus. On this basis, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for embryo-foetal development were considered to be ≥ 1000 mg/kg bw/day.
Executive summary:

In a developmental toxicity study performed according to OECD TG No. 404 and in accordance with GLP, the test substance was administered to 22 time-mated female CD rats via oral gavage administration at dose levels of 0, 100, 300 or 1000 mg/kg bw/day at a dose volume of 10 mL/kg bodyweight from days 6 through 19 of gestation. The dose levels in this study were based on the results of an OECD 407 and an OECD 415 studies in CD rats.

Body weights, food intake and clinical observations were recorded. The animals were killed on Day 20 of gestation, a necropsy performed, and the internal organs examined for gross abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, the sex determined and examined for external, visceral, skeletal and cartilage abnormalities.

There were no test item-related deaths or clinical observations in pregnant female rats that were related to the test item administration at any dose level.

Following the first day of administration on Day 6 of gestation, in the group given 1000 mg/kg bw/day, less food was eaten, accompanied by a slight but statistically significant mean body weight loss. In the group given 300 mg/kg bw/day, mean body weight gain was also slightly lower than Controls after the first day of administration on Day 6 of gestation. From Day 7 of

gestation, however, food intake and mean body weight gain in all groups were similar to, or in excess of Controls, such that by Day 20 of gestation, all body weight values were similar across

the groups, and mean gravid uterus weight when adjusted for body weight was comparable with Controls.

On Day 20 of gestation, there were 20, 21, 21 or 20 females pregnant with live foetuses in the groups given 0, 100, 300 or 1000 mg/kg bw/day, respectively. There was no effect of the test item on any of the pregnancy parameters.

There were no foetal abnormalities that were considered related to the test item administration.

Administration of the test item to the pregnant Crl:CD(SD) rat at 100, 300 or 1000 mg/kg bw/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, was generally well tolerated at all dose levels. A mild and transient effect on maternal food consumption and weight gain was observed over the first day of administration on Day 6 of gestation, with no effect on the developing conceptus or foetus. On this basis, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for embryo-foetal development were considered to be ≥ 1000 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 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

A key study was identified (Sequani, 2018, rel.1). In this developmental toxicity study performed according to OECD TG No. 404 and in accordance with GLP, the test substance was administered to female CD rats via oral gavage administration at dose levels of 0, 100, 300 or 1000 mg/kg bw/day at a dose volume of 10 mL/kg bodyweight from days 6 through 19 of gestation.

There were no test item-related deaths or clinical observations in pregnant female rats that were related to the test item administration at any dose level.

Following the first day of administration on Day 6 of gestation, in the group given 1000 mg/kg bw/day, less food was eaten, accompanied by a slight but statistically significant mean body weight loss. In the group given 300 mg/kg bw/day, mean body weight gain was also slightly lower than Controls after the first day of administration on Day 6 of gestation. From Day 7 of gestation, however, food intake and mean body weight gain in all groups were similar to, or in excess of Controls, such that by Day 20 of gestation, all body weight values were similar across the groups, and mean gravid uterus weight when adjusted for body weight was comparable with Controls.

On Day 20 of gestation, there were 20, 21, 21 or 20 females pregnant with live foetuses in the groups given 0, 100, 300 or 1000 mg/kg bw/day, respectively. There was no effect of the test item on any of the pregnancy parameters.

There were no foetal abnormalities that were considered related to the test item administration.

Administration of the test item to the pregnant Crl:CD(SD) rat at 100, 300 or 1000 mg/kg bw/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, was generally well tolerated at all dose levels. A mild and transient effect on maternal food consumption and weight gain was observed over the first day of administration on Day 6 of gestation, with no effect on the developing conceptus or foetus. On this basis, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for embryo-foetal development were considered to be ≥ 1000 mg/kg bw/day.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008 (CLP).

Self-classification:

Based on the available information, no self-classification is proposed according to the CLP or the GHS.