Reaction mass of (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl palmitate and (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl stearate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The results of a reproductive/developmental toxicity screening study (OECD 421) with the test item in rats by the oral route indicate no potential to impair fertility. The NOAEL for adult fertility and reproduction parameters is set to the highest dose tested (1000 mg/kg bw/day). 

No classification is warranted under Regulation (EC) 1272/2008.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Hannover rat is the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 200 to 225 g for males and 175 to 200 g for females
- Housing:
The animals were housed in a limited access rodent facility. From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate,Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.

- Diet (e.g. ad libitum): ad libitum (laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4,
20019, SettimoMilanese (MI), Italy)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01.03.2021 To: 14.04.2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test item was suspended in the vehicle. The preparations were made daily at concentration of 25, 75 and 250 mg/mL, unless specified otherwise based on stability data stated in the validation study (section 3.4). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1 (one exception since one low dose male was found dead before the start of the mating phase)
- Length of cohabitation: until positive identification occured
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations (ERBC Study no. A4158). The analysis confirmed a 11 days stability at room temperature in the range from 25 to 250 mg/mL.
Samples of the preparations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the homogeneity and concentration.
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC. The validated software used for this activity was Analyst 1.6.2. Results of the analyseswere within the acceptability limits stated in ERBC SOPs for concentration of suspension (85-115%),

Duration of treatment / exposure:

- Males: at least 28 days. The duration of treatment covered a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.

- Females: up to 60 days. The duration of treatment covered a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice.

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

Test group 1 (Control)

Dose / conc.:

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

Remarks:

Test group 2

Dose / conc.:

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

Remarks:

Test group 3

Dose / conc.:

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

Remarks:

Test group 4

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels have been selected in consultation with the Sponsor based on information from previous studies.
In a previous repeated-dose toxicity study (OECD 407) with a structural similar substance the NOAEL was considered to be 1000 mg/kg/day

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Mortality: Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
- Clinical observations: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: once a week
- Males: weekly from allocation to termination.
- Females: at weekly interval from allocation, where possible, to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
- Dams were weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.
FOOD CONSUMPTION: Yes
- Time schedule: weekly during the pre-mating period, starting Day 1 of dosing up to mating.
- Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum
WATER CONSUMPTION (if drinking water study): No
CLINICAL BIOCHEMISTRY: Yes
- Anaesthetic used for blood collection (for hormone determination) : Yes (Isoflurane)
- How many animals: all parental males and females/ Pups: from each litter (1 sample for males and 1 sample for females)
- Parameter checked: T4, TSH (serum)
REPRODUCTIVE PERFORMANCE
- Mating: 1:1 (with one exception since one low dose male was found dead before the start of the mating phase)
- Parturition and gestation length: A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition was complete) was defined as Day 0 post partum.

Oestrous cyclicity (parental animals):

- Oestrus cycle was monitored by vaginal smears for at least 2 weeks before allocation.

Sperm parameters (parental animals):

Spermatogenic cycle was examined.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum
- maximum of 4 pups/sex/litter

PARAMETERS EXAMINED
- Pup identification: As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
- Pup weight: Live pups were individually weighed on Days 1, 4 and 13 post partum.
- Pup observation: performed once daily for all litters.
- Clinical biochemistry: Blood collection for hormone determination (T4, TSH in serum) was performed only for animals at termination. On Days 4 and 14 post partum, as part of the necropsy procedure, blood samples of approximately 0.5 mL (by sex) were taken from each litter (1 sample for males and 1 sample for females, when possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture).Anogenital distance (AGD): measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.
- Nipple count on Day 13 post partum: No nipples/areolae in male pups were recorded. These data were not tabulated but taken and archived with all study raw data.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
Parental animals that had completed the scheduled test period were killed by exsanguinations under isoflurane anaesthesia. One female of the low dose group was sacrificed for humane reasons, before the scheduled test period, under carbon dioxide asphyxiation.
The males were killed at completion of mating of all females (after 35 days of treatment). The females with live pups were killed on Day 14 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. One control female did not show evidence of copulation, however it gave birth. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

GROSS NECROPSY
- Females: All females were also examined for the number of visible implantation sites (pregnant
animals). Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS
- Parental animals: From all animals completing the scheduled test period, the following organs/tissues were dissected free of fat and weighed: Abnormalities, epididymides, liver, ovaries with oviducts, prostate gland (dorsolateral and ventral), seminal vesicles with coagulating glands, testes, thyroid gland, uterus – cervix, vagina.
- The ratios of organ weight to body weight was calculated for each animal.

TISSUE FIXATION
Samples from all the tissues/organs mentioned under “organ weights” were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, Harderian glands, ovaries, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol)

HISTOPATHOLOGY
- The following tissues were required for histopathological examination: Abnormalities, epididymides, liver, ovaries with oviducts, prostate gland (dorsolateral and ventral), seminal vesicles with coagulating glands, testes, thyroid gland, uterus – cervix, vagina.
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
- The examination was restricted as described:
o Tissues mentioned in “organ weights” from all males and females in the control and high dose groups killed at term. The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also included.
o Tissues mentioned in “organ weights” from all animals killed or dying during the treatment period.
o All abnormalities in all groups.
- The examination of liver and thyroid was extended to animals of the low and mid-dose groups of both sexes based on treatment-related changes observed in the high dose group.

Postmortem examinations (offspring):

SACRIFICE
Pups killed for humane reasons or those that had completed the scheduled test period (Day 4 or Day 14 post partum) were euthanised by intraperitoneal injection of Thiopenthal. Pups selected for blood collection for hormone determination were killed on the day of blood sampling.

GROSS NECROPSY
- Pups – unscheduled dead: All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities
- Pups – Day 4 post partum: All culled pups sacrificed at Day 4 post partum were subjected to an external examination.
- Pups – Day 14 post partum: All live pups sacrificed on Day 14 post partum were examined for external abnormalities. Gonads were inspected from all pups in order to confirm the sex previously determined by external examination.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

Male mating index (%) = no. of males with confirmed mating/ no. of males cohabitated x 100
Male fertility index (%) = no.of males which induced pregnancy/ no.of males cohabitated x 100
Female mating index (%) = no. of females with confirmed mating/ no. of females cohabitated x 100
Female fertility index (%) = no.of pregnant females / no.of females cohabitated x 100

Males and Females Precoital Interval = The number of nights paired prior to the detection of mating
Prenatal loss (%) = (no.of visible implantations − live litter size at birth) / no.of visible implantations x 100

Offspring viability indices:

Postnatal loss at Day 4 post partum (%) = (Live litter size at birth - live litter size at Day 4 (before culling)/ Live litter size at birth x 100

Postnatal loss at Day 13 post partum (%) = (Live litter size on Day 4 (after culling) - Live litter size on Day 13)/ Live litter size on Day 4 (after culling) x 100

Pup loss at Day 0 post partum (%) = (Total litter size - Live litter size)/ Total litter size x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs were recorded in males throughout the study.
No treatment-related clinical signs were recorded in females before pairing. During gestation, signs of difficulty to delivery were recorded in one female at 100 mg/kg/day.
No treatment-related clinical signs were recorded during post partum phase.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On gestation day 22, one female at 100 mg/kg/day was sacrificed following the occurrence of important clinical signs like prolapse of the uterus, pallor, piloerection and coldness to touch. The presence of these signs was related to difficulty in delivery.
One female at 300 mg/kg/day was found dead on gestation day 23. This female gave birth 8 pups on gestation day 22 (found dead) and additional 3 live pups were found in the cage on Day 23 post coitum. Both females had a prolonged time of gestation without completing the parturition and the impaired delivery was the reason of the humane sacrifice for the low dose female (100 mg/kg/day) and the occurrence of premature death for the mid-dose female (300 mg/kg/day). The gross and microscopic evaluation did not allow to establish the cause of impaired parturition. However, since these findings occurred in single animals, they could be considered unrelated to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

A dose-related increase in the TSH level was observed in parental males. The increase was 63% at 300 mg/kg/day and 113% at 1000 mg/kg/day, compared to controls. This finding could be related with the follicular cell hypertrophy of thyroid gland observed at histopathology and, due to the absence of T4 changes, could represent a subclinical hypothyroidism. Table included under "Any other information on results incl. tables".

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic observations associated with the oral administration of the test item were present in the thyroid and liver.
- In the thyroid gland, treatment-related microscopic observations consisted in follicular cell hypertrophy which was characterised by the presence of increased size and height of follicular cells with diffuse distribution. Follicular lumen was decreased in size and filled with normal staining to pale colloid and the cytoplasm of the follicular cells was enriched with small clear vacuoles. The above mentioned finding was mild to marked in all males (10/10) and moderate in one female (1/10) of high dose group and of mild severity in males (3/10) of mid-dose group. In males at the mid and high dose, this finding correlated with
changes in thyroid related hormone levels measured in parental males (increased values of TSH) and with the increased absolute and relative thyroid mean weights. Follicular cell hypertrophy of the thyroid gland was considered treatment-related.
- In the liver, treatment-related microscopic observations consisted in centrilobular hepatocellular hypertrophy of
minimal to mild severity was present in all males (10/10) and most females (7/10) at the high
dose and of minimal severity in few males (2/10) of the mid-dose group. Hepatocyte hypertrophy
consisted of enlarged hepatocytes containing variable amounts of fine granular pale
eosinophilic (ground glass) cytoplasm.
- There were no microscopic observations in the
testes as per specific stage aware evaluation using PAS stain.
- Any microscopic observations other than those listed above had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

- During pairing, the mating performance i.e. the number of days paired to spermpositive
days (pre-coital interval) was similar between groups. The resulting reproductive parameters,
in terms of copulatory and fertility indices, did not show any differences between control and treated groups
- The gestation length was similar in all groups. The number of implantation sites did not show significant differences between groups as well as the pre-natal loss.
- The litter size at birth, on Day 1 post partum (before culling) and on Day 13 post partum
(after culling) did not show differences between control and treated groups. The mean litter and pup weights at birth, on Days 1, 4 and 13 post partum was comparable
between groups.

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: the relationship between thyroid hypertrophy and TSH higher level is not necessarily indicative of thyroid dysfunction since the thyroid hormone changes did not include low serum of T4

Dose descriptor:

NOAEL

Remarks:

(Reproduction)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The observation of pups did not reveal treatment-related findings. Pale or small appearance, the presence of wound in the muzzle, not opened eye, or tip of tail missing were seen as single occurrences regardless the treatment groups.

Clinical biochemistry findings:

no effects observed

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distance (normalized to the cube root of the body weigh on Day 1 post partum) did not differ in male pups of treated groups when compared to controls whereas it was significantly increased in females of all treated groups (up to 28%, respect to the control group).
Although a dose relation was evident, the statistical differences were not considered of toxicological significance but rather due to unexpected lower control, being all values in the range of the historical control data (HCD). Table included under "Any other information on results incl. tables".

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes were seen in thyroid weight from male and female pups of treated
groups compared to controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Findings at necropsy included the absence of milk in the stomach from pups sacrificed for
humane kill. All autolysed organs were frequently found in decedent pups.
The necropsy of culled pups (Day 4 post partum) and those sacrificed at termination (Day 14 post partum) did not show treatment-related changes. For those pups found dead, the organ were autolysed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Table 1: Liver and thyorid weights

		Males				HCD	Females				HCD
Dose level (mg/kg/day)		Control	100	300	1000		Control	100	300	1000
Liver	Abs. weight (g)	12.282	14.248*	14.946*	16.092*	14.52	12.234	12.608	13.924	15.227*	11.29
	Rel. weight (g)	2.798	3.210*	3.350*	3.636*	3.65	3.955	4.221	4.534	4.854*	3.83
Thyroid	Abs. weight (g)	0.0230	0.0254	0.0284*	0.0283*	0.026	0.0221	0.0216	0.0238	0.0230	0.023
	Rel. weight (g)	0.0053	0.0057	0.0064	0.0064	0.006	0.0074	0.0072	0.0078	0.0074	0.008
* = Statistical significant from control at p<0.05

 

Table 2: Thyroid hormone measurements in parental males and male and female pups

Parental Males (Group mean data)
		Group
Parameter/units		1	2	3	4
Thyroxine nmol/L	Mean SD N	41.4 3.9 10	41.4 3.6 9	44.8 5.3 10	38.1 4.1 10
Thyroid Stimulating Hormone ng/mL	Mean SD N	8.79 1.99 10	9.6 1 1.74 9	14.34+ 4.24 10	18.68+ 9.13 10
Controls from group(s): 1                           Subgroup(s): 1 * = mean value of group is significantly different from control at p < 0.05 + = mean value of group is significantly different from control at p < 0.01
Pups Day14 Post Partum_Males (Group mean data)
		Group
Parameter/units		1	2	3	4
Thyroxine nmol/L	Mean SD N	54.9 10.1 7	52.3 6.5 7	52.5 6.9 8	53.3 2.8 9
Thyroid Stimulating Hormone ng/mL	Mean SD N	3.80 0.65 7	3.60 0.68 7	3.83 0.72 8	3.67 0.85 9
Controls from group(s): 1                           Subgroup(s): 1 * = mean value of group is significantly different from control at p < 0.05 + = mean value of group is significantly different from control at p < 0.01
Pups Day14 Post Partum_Females (Group mean data)
		Group
Parameter/units		1	2	3	4
Thyroxine nmol/L	Mean SD N	56.0 7.6 7	55.3 10.5 7	55.6 8.9 8	54.8 8.0 9
Thyroid Stimulating Hormone ng/mL	Mean SD N	3.54 0.53 7	3.71 0.53 7	4.33 0.89 8	3.67 0.76 9
Controls from group(s): 1                           Subgroup(s): 1 * = mean value of group is significantly different from control at p < 0.05 + = mean value of group is significantly different from control at p < 0.01

 

Table 3 Anogenital distance

Females_ANOGENITAL DISTANCE ON DAY 1 POST PARTUM – GROUP MEAN DATA
Anogenital Distance (normalized) mmg/g^1/3)^
	Group
	1	2	3	4	HCD
Mean SD N	1.07 0.12 7	1.28* 0.17 7	1.33* 0.12 8	1.37* 0.05 9	1.39
^ = normalised to the cube root of the body weight collected on Day 1 post partum (mm/g) Statistical analysis: Kruskall Wallis test T test if group mean differences are different from control at p < 0.05 * = mean value of group is significantly different from control
Males_ANOGENITAL DISTANCE ON DAY 1 POST PARTUM – GROUP MEAN DATA
Anogenital Distance (normalized) mmg/g^1/3)^
	Group
	1	2	3	4	HCD
Mean SD N	2.52 0.16 7	2.50 0.10 7	2.62 0.18 8	2.47 0.17 9	2.06
^ = normalised to the cube root of the body weight collected on Day 1 post partum (mm/g) Statistical analysis: Kruskall Wallis test T test if group mean differences are different from control at p < 0.05 * = mean value of group is significantly different from control

 

Table 4 Historical Control Data of the Anogenital distance

Anogenital Distance- Historical Control Data
	Mean mmg/g^1/3	Max mmg/g^1/3	Min mmg/g^1/3	N
AGD (mmg/g^1/3) Male Pups	2.06	2.96	1.05	183	Pups
AGD (mmg/g^1/3) Female Pups	1.39	8.19	0.55	155	Pups
Average study
AVG_study AGD (mmg/g^1/3) Male Pups	2.05	2.26	1.94	3	studies
AVG_study AGD (mmg/g^1/3) Male Pups	1.35	1.85	0.93	3	studies

Conclusions:

In conclusion after repeated dose, treatment-related effects were seen in parental animals at 300 and 1000 mg/kg/day characterized by morphological changes in thyroid and liver tissues (hypertrophy) as well as by TSH alteration in males (increase). Nevertheless, the relationship between thyroid hypertrophy and TSH higher level is not necessarily indicative of thyroid dysfunction since the thyroid hormone changes did not include low serum of T4. No adverse effects were observed regarding developmental and reproductive parameters.
Based on these results, the NOAEL (No Observed Adverse Effect Level) can be conservatively
set at 100mg/kg/day for general repeated dose toxicity. However, no clear indications of
adverse effects were seen at the higher dose levels.
The NOAEL can be set at 1000mg/kg/day for reproduction/developmental toxicity.

Executive summary:

General and reproductive/developmental toxicity of the test item, according to the OECD guideline 421, was investigated in Wistar Hannover rats. The study was conducted to provide information on systemic toxicity as well as effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring up to Day 14 post partum.
The animals were administered orally by gavage at 0 (vehicle control) 100, 300 and 1000 mg/kg/day. The vehicle for dosing control animals and for dose preparations was corn oil and the dose volume was set at 4mL/kg body weight.
General toxicity
- Assessment of in-vivo parameters of body weight/body weight gain, food consumption and daily clinical observation did not show differences between control and treated groups throughout the study, both in male and female animals.
- A dose related increase of circulating levels of TSH was observed in males whereas T4 was comparable between groups.
- No treatment-related macroscopic observations were recorded at the end of the treatment period.
- Liver and thyroid weights (absolute or relative to body weight) were increased at all dose levels compared to the control group and the organ weight change magnitude was also proportional to the dose. These changes correlated with the histopathological findings in thyroid and liver of high dose animals (both sexes) and of the mid-dose males, consisting in hepatocytic hypertrophy for liver and follicular cell hypertrophy for thyroid.
- The evaluation of the spermatogenic cycle did not indicate any difference between control and high dose males.

Reproductive and developmental toxicity
- Concerning the reproductive parameters no differences were seen in the mating performance including the pre-coital interval (i.e. the number of days paired to sperm positive day) and the copulatory evidence (i.e. the positive identification of mating verified by the presence of sperm and/or copulation plug in situ or in the cage tray).
- The resulting copulatory and fertility indices, did not show any differences between control and treated groups. All dams had comparable length of gestation and gave birth on Days 22/23 post coitum as mean value.
- The developmental landmarks including the litter size, mean pup and litter weights were comparable between groups. The offspring did not show treatment-related clinical signs.
- In addition, hormones determination of circulating TSH and T4 did not differ between control and treated groups as well as the thyroid weights in pups on Day 14 of age.
- No adverse effects were seen in the anogenital distance both in males and female pups of treated groups, when compared to controls.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive/developmental toxicity screening test (OECD 421): 

General and reproductive/developmental toxicity of the test substance, according to the OECD guideline 421, was investigated in Wistar Hannover rats. The study was conducted to provide information on systemic toxicity as well as effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring up to Day 14 post partum. The test substance was administered daily by oral gavage to male (up to 35 days) and female (up to 60 days) Wistar Hannover rats. The vehicle was corn oil and the dose volume was set at 4 mL/kg bw. Dose levels (100, 300, 1000 mg/kg bw/day) have been selected based on information from previous studies.

No difference between control and treated groups in both sexes was observed for body weight/ body weight gain, food consumption and clinical observation. Effects observed refer to changes in the liver, thyroid and hormone levels of parental animals. Increased organ weight changes were noted in the thyroid (mid and high dose males) and the liver (all doses in males and in high dose females) and correlated with microscopic changes (thyroid: follicular cell hypertrophy; liver: hepatocytic hypertrophy). In addition, dose related increase of circulating levels of TSH was observed in males only. T4 values were comparable between groups. The NOAEL (No Observed Adverse Effect Level) was conservatively set at 100 mg/kg/day for general repeated dose toxicity.

No differences were observed for reproductive parameters including oestrous cycle, pre-coital intervals, copulatory index, fertility index, gestation length, number of implantation sites, prenatal loss and spermatogenic cycle. Developmental landmarks including litter size (birth, Day 1 & 13 post partum), mean litter and pup weight (birth, Day 1, 4, 13 post partum) were comparable between groups and no treatment-related clinical signs were observed in pups. No differences between control and treated groups were observed concerning thyroid hormone (TSH, T4) and thyroid weight (PND 14 pus) measurements. No adverse effects were seen in the anogenital distance. The NOAEL was set at 1000 mg/kg/day for reproduction/developmental toxicity

Effects observed for thyroid and liver tissue, were also in consistency with effects observed in an additional 28-day repeated dose study (RCC.848428.2003). This study was conducted with a structural analogue in male and female rats (Source substance, CAS 376588-17-9) and dose levels were set to 50 (low dose), 200 (mid dose) and 1000 (high dose) mg/kg bw/day. Increased liver weight changes (absolute: mid and high dose females and high dose males) also correlated with an increased incidence of a minimal to slight hepatocellular hypertrophy (high dose animals). However, after the recovery period this change reverted to normal levels. It was regarded as an adaptive change most likely caused by the metabolic biotransformation of the test item. Further, this change was accompanied by an increased gamma glutamyl transferase activity after four weeks treatment (mid and high dose females and high dose males) when compared with the controls. This finding was also reversible after two weeks recovery. In contrast to the OECD 421, no change in the thyroid weight was noted. However, a hypertrophy of the follicular epithelium was also recorded (high dose males). After the recovery period, the incidence of follicular hypertrophy was largely reduced. All other parameters examined in the 90-day study including mortality, clinical signs, functional observational battery, food consumption, hematology, urinalysis, clinical biochemistry (except the increased gamma glutamyl transferase activity) were not affected or at least not of toxicological relevance.

Overall, increased liver weight coupled with hepatocellular hypertrophy and additional clinical chemistry changes (28-day study: gamma glutamyl transferase activity) is considered evidence of an increased metabolic capacity due to xenobiotic exposure. Observed thyroid changes are considered to be a secondary effect due to this increased hepatocellular metabolic capacity. This mode of action is well described in detail in several peer-reviewed publications (Meek et al., 2003; McClain, 1989; Zoeller et al., 2007). Furthermore, the rat shows a particularly high sensitivity to chemicals which act via this mode of action, whereas humans are far less sensitive (Meek et al., 2003; McClain, 1989). The reason for this are widely accepted species differences in the thyroid hormone regulation. These comprise e.g. the lack of the thyroxine-binding protein in the adult rat in combination with shorter half-lives of T4 and T3. Altogether, the effects observed on thyroid hormone changes are considered secondary to liver enzyme induction and due to species differences, these findings are regarded as not relevant for humans. Therefore, classification is not warranted under Regulation (EC) 1272/2008.

 

Meek, M. E. & et al. A Framework for Human Relevance Analysis of Information on Carcinogenic Modes of Action. Crit. Rev. Toxicol. 33, 591–653 (2003).

McClain, R. M. The Significance of Hepatic Microsomal Enzyme Induction and Altered Thyroid Function in Rats: Implications for Thyroid Gland Neoplasia. Toxicol. Pathol. 17, 294–306 (1989).

Zoeller, R. & et al. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit. Rev. Toxicol. 37, 11–53 (2007).

Effects on developmental toxicity

Description of key information

The results of a reproductive/developmental toxicity screening study (OECD 421) with the test item in rats by the oral route indicate no potential to impair development. The NOAEL for adult development is set to the highest dose tested (1000 mg/kg bw/day). 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Reproductive/developmental toxicity screening test (OECD 421): 

General and reproductive/developmental toxicity of the test substance, according to the OECD guideline 421, was investigated in Wistar Hannover rats. The study was conducted to provide information on systemic toxicity as well as effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring up to Day 14 post partum. The test substance was administered daily by oral gavage to male (up to 35 days) and female (up to 60 days) Wistar Hannover rats. The vehicle was corn oil and the dose volume was set at 4 mL/kg bw. Dose levels (100, 300, 1000 mg/kg bw/day) have been selected based on information from previous studies.

Developmental landmarks including litter size (birth, Day 1 & 13 post partum), mean litter and pup weight (birth, Day 1, 4, 13 post partum) were comparable between groups and no treatment-related clinical signs were observed in pups. No differences between control and treated groups were observed concerning thyroid hormone (TSH, T4) and thyroid weight (PND 14 pus) measurements. No adverse effects were seen in the anogenital distance. The NOAEL was set at 1000 mg/kg/day for reproduction/developmental toxicity

Classification for developmental toxicity is not warranted under Regulation (EC) 1272/2008.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008.