Sulfonic acids, C6-8-alkane, sodium salts

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

An extended one generation reproductive toxicity study has not been performed on this substance. In a 90 -day repeated dose toxicity study and in an extended 14 -day repeated dose range finding study there were no effects noted in reproductive organs related to test item administration at any dose level. On the basis of these studies, the substance is not expected to cause adverse effects on fertility.

A screening test was not performed because a prenatal developmental toxicity study is available for this substance.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An extended one generation reproductive toxicity study has not been performed on this substance. In a 90 -day repeated dose toxicity study and in an extended 14 -day repeated dose range finding study there were no effects noted in reproductive organs related to test item administration at any dose level. On the basis of these studies, the substance is not expected to cause adverse effects on fertility.

A screening test was not performed because a prenatal developmental toxicity study is available for this substance.

Effects on developmental toxicity

Description of key information

OECD TG 414 performed using the registered substance, Alkane C6-C8 (even numbered), 1-sulphonic acid, sodium salt

NOAELmaternal toxicity: 1000 mg a.i./kg bw/day, based on no maternal systemic effects at 1000 mg a.i./kg bw/day.

NOAELembryotoxicity: 1000 mg a.i./kg bw/day, based on the lack of any test-item related intra-uterine effect in any treatment group.

NOAELfoetotoxicity: 1000 mg a.i./kg bw/day, based on no effect on runts at 1000 mg a.i./kg bw/day.

NOAELteratogenicity: 1000 mg a.i./kg bw/day, based on the lack of treatment related malformations in any dose group.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2019 - 12 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch/Lot number: 0008282300
Description: Clear liquid
Purity: 40.9 % in water
Expiry date: 13 July 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity)

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar rats CRL:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, 13-14 weeks old at mating.
- Weight at study initiation: 218 - 289 g (the variation did not exceed ± 20% of the mean weight).
- Fasting period before study: none
- Housing: Type II polycarbonate cages were used during mating and gestation period and Type III polycarbonate cages were used during the acclimatisation period. Successfully mated animals were housed individually.
- Diet (e.g. ad libitum): ssniff® SM "Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance" (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): tap water (in water bottles) as for human consumption, ad libitum.
- Acclimation period: At least 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-24.7°C (target: 22 ± 3°C).
- Humidity (%): 22-56% (target: 30-70%).
- Air changes (per hr): 15-20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 11 November 2020 To: 12 December 2020

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of Charles River Laboratories Hungary Kft.
Formulations were prepared prior to administration to the animals within the stability parameters established via the analytical method development study of the Test Site 1 for analytical work. Based on these results, the test item is stable in the vehicle for at least ten days at room temperature.
Stability of the test item in the vehicle was assessed under the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to Test Site cross-validated method development study).
A correction factor of 2.45 was used at dose formulation preparation to take account of the active substance content in the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor and the results of a trial formulation as well as a Dose Range Finding (DRF) study, distilled water was selected as a suitable vehicle for this study
- Lot/batch no. (if required): Supplier: Magilab Kft.; Lot No.: 201909057; Expiry date: 20 March 2020; Storage conditions: Room temperature
- Dose volume: 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration was performed at the Test Site 1 for analytical work using a developed analytical method (19/211-901ANE). The test item concentration was analysed By Fumoprep Kft. using a HPLC-MS method. Duplicate samples were taken from the test item formulations at least on 2 occasions during the study (13 November 2019 and 04 December 2019) one set for analysis and one set as a back-up, if required for any confirmatory analyses. Duplicate samples were similarly taken from the vehicle control formulation for analysis.
Acceptance criterion of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 15% of the nominal concentration. Formulation samples were kept at room temperature until shipment. Samples to be analysed were shipped to the Principal Investigator 1 (PI 1) as soon as practical after collection for concentration measurements.
A summary of the analysis results obtained are presented in the results section below (Table 1).
Detailed results from each sampling point are attached.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1male:1 female
- Length of cohabitation: Daily for 2-3 hours until 24 sperm positive females/group were attained
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as [day 0] of pregnancy

Duration of treatment / exposure:

gestation day 6 (GD 6) to gestation day 19 (GD 19)

Frequency of treatment:

Daily

Duration of test:

GD 0 - GD 20

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

based on active ingredient

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

based on active ingredient

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

based on active ingredient

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

No. of animals per sex per dose:

24 mated females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. A computer software program (Provantis v9) was used to verify homogeneity/variation among/within groups.
- Fasting period before blood sampling for (rat) dam thyroid hormones: For thyroid hormone analysis, blood samples were taken from all dams at termination by venepuncture into tubes containing K3-EDTA as anticoagulant

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: GD 6, then weekly

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20
- Body weight gains were calculated for each interval, including GD 0-6, GD 6-20, GD 0-20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Food was measured on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20
- Food consumtpion was calculated for each interval, including GD 0-6, GD 6-8, GD 8-10, GD 6-20 and GD 0-20

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes

OTHER: The weight of the thyroid gland with parathyroid glands for all dams were measured with a precision of at least 0.001g (as a paired organ, they were weighed together). Absolute organ weights were measured, and organ weights relative to the body weights were calculated and reported. If any significant difference in size is noted between paired organs, an individual weight of each organ was 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
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Blood sampling:

- Plasma: Yes
- Serum: No
- Volume collected : 125 μL for the first aliquot and at least 75 μL for the second aliquot

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: yes

Statistics:

Statistical evaluation of data was performed with SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 for data tabulated in Excel.
For SAS v9.2 (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova /Ancova (one-way analysis of variance) test was carried out. If the result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of intergroup differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. For non-continuous data, the Cochran-Amitage test for trend was applied and the Chisquared test was used for statistical differences relative to control.
Non-pregnant females, females with no implantation or ≤ 5 implantation sites, and females showing signs of abnormal pathology and/or mis-dosing were excluded from selected statistical analysis; in-life individual data was reported as applicable.
The limit for growth-retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded (runt) if the deviation from the mean control values was greater than minus two-fold standard deviation of all control foetuses (2.915 g for males and 2.869 g for females.).

Historical control data:

Attached (Appendix 9).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One High dose female (4521) was found dead on Day 10. Decreased activity, piloerection, hunched back, tremor, red left eye/nose were clinically recorded for this female. This death was considered to be the result of a local effect of the test item. Occasional deaths, due to reflux or similar respiratory exposure, after oral dosing with surfactants may be anticipated.
Thin fur was observed in 1 out of 23 animals in the High dose group.
Piloerection was present in 1 out of 24 animals in the Mid dose group, and 3 out of 23 animals in the High dose group. The finding appeared from Day 9 until Day 16, the symptom persisted for a maximum of six days.
Laboured respiration was observed in 1 out of 23 animals in the High dose group.
Slight noisy respiration was present in 4 out of 23 animals in the High dose group. The finding appeared from Day 8 until Day 19.
Noisy respiration is a common local effect with surfactants because even a very small amount entering the trachea (e.g. from reflux) may cause local irritation.
All other changes were considered incidental, and not thought to be test item related effects.
Summary tables from the study report are attached.

Mortality:

mortality observed, treatment-related

Description (incidence):

One High dose female (4521) was found dead on Day 10. Decreased activity, piloerection, hunched back, tremor, red left eye/nose were clinically recorded for this female. This death was considered to be the result of a local effect of the test item. Occasional deaths, due to reflux or similar respiratory exposure, after oral dosing with surfactants may be anticipated.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related body weight effects were observed in any of the dose groups.
No significant dose-related effects on corrected body weight, corrected body weight gain, and net body weight gain were observed.
Selected body weight data are shown in Table 2.
Detailed body weight data of the evaluated animals are attached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A transient decrease of food consumption in the High dose group was observed at the start of the treatment.
Food consumption data of the evaluated animals are attached.

Endocrine findings:

no effects observed

Description (incidence and severity):

Blood samples were taken from all dams at termination and 95 blood samples were successfully analysed. There were no statistically significant changes in the concentration of the T3, T4 and TSH level between the Control and the Dose groups.
Summary tables are attached.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

During the macroscopic examination of dams, the thyroid gland with parathyroid glands were retained from all animals and the organ weights recorded. There were no statistical differences in the organ weight between the Control and the Dose groups.
Summary data are attached.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dark/red discoloration of the non-collapsed lungs, seen in the one found dead female, (4521) may have been treatment related although it is a common agonal/post-mortem change; enlarged adrenal glands were also observed macroscopically. There was no evidence for clear systemic toxicity in the dead animal.
For pregnant females, no evidence of test item-related macroscopic and microscopic changes were observed in animals dosed at 100, 300 and 1000 mg a.i./kg bw/day on necropsy Day 20.
There were no gross changes observed in the one non-pregnant Low dose female (2506) on necropsy Day 20.
Summary tables are attached.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. There were no statistical differences between the Control and the Dose groups.
Summary data are attached.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the pre- and post-implantation loss in the test item treated animals when compared to the control.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no significant differences in early or late embyronic loss between controls and any test item group.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses observed in any group.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Ninety-six females (24 each for the Control, the Low, Mid, and High dose group, respectively) were mated in the study. The number of confirmed pregnant, evaluated dams was: 24 in the Control and the Mid dose group and 23 each in the Low and High dose groups. See table 3 below.

Other effects:

no effects observed

Description (incidence and severity):

No placental abnormalities were observed in any of the control or test group animals.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: no maternal systemic effects at the high dose

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant change in the body weights were observed in animals in the Low, Mid, and High dose groups. All values were within the historical control range (mean ± SD: 3.407 ± 0.225).
There was no significant change in the number of foetuses with retarded body weight in any of the dose groups

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no significant differences in the sex distribution (%males/females) between controls and any of the test groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no significant differences in the mean litter size between controls and any of the test groups.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no significant differences in anogenital difference for males, females or combined males/females between the controls and any of the test groups.

External malformations:

no effects observed

Description (incidence and severity):

No external variations or malformations were seen in the test item treated groups. Refer to table 6 below.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The observed skeletal abnormalities are summarized in Table 9 below. Most of the skeletal findings were within the current historical control range or the concurrent study control data or were considered to be incidental findings without dose response (see table 10, attached, for detailed data).
Analysis of the skeletal findings showed the numbers of malformed / intact foetuses were comparable with the control in the Low, Mid and High dose groups. The number of variations were higher in all test item treated groups.
For the majority of the skeletal variations, the foetal or litter based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as not biologically significant and not related to test item treatment.
Most of the findings were within the study control data ranges or had isolated occurrences that were considered incidental and ascribed to individual variability.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The observed visceral abnormalities are summarized in Table 7. Most of the findings were within the current historical control ranges or the study control data or had an isolated occurrence that was considered incidental, ascribed to individual variability and therefore not related to treatment. (see table 8 attached for detailed data)

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no test-item related intra-uterine effect in any treatment group

Dose descriptor:

NOAEL

Remarks:

foetotoxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effect on runts at 1000 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no treatment related malformations in any dose group

Developmental effects observed:

no

Table 1: Summary of the dose formulation analysis

Formulation	13 November 2019		04 December 2019
	Mean measured concentration (mg/L)	Relative to the nominal concentration (%)	Mean measured concentration (mg/L)	Relative to the nominal concentration (%)
Control	not detected	-	not detected	-
20 mg a.i./mL	20.64 ± 0.39	103.0	19.16 ± 0.2	95.6
60 mg a.i./mL	64.52 ± 1.76	107.3	58.11 ± 0.98	96.6
200 mg a.i./mL	215.0 ± 3.41	107.3	187.3 ± 1.89	93.4

Note: Samples were collected freshly on the days indicated in the header of the table.

Samples for test item concentration determination of the dosing formulations were collected twice during the treatment period. Formulation stability was established before

this study. The test item concentration was analysed By Fumoprep Kft. using a HPLCMS method.

The mean concentration of all formulations was found to be in the range of 93.4-107.3% of their nominal concentrations (20, 60, and 200 mg/mL) and were found

to be homogenous.

No test item was detected in the vehicle control formulations.

Based on these results, test item formulations were considered suitable for the study purposes.

Table 2: Selected body weight parameters

Parameters	Historical control	Dose (mg a.i./kg bw/day)
		0	100	300	1000
Number of evaluated dams	507	24	23	24	23
Body weight gain GD0 -3 (g)	10.9±4.2	10.4	9.5	12.9##	9.9

Notes: Historical control data is mean ± 1SD. Body weight data were rounded to one decimal place.

Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.

##= p<0.05; Dunnett two sided test

Table 3: Summary of pregnancy data

Parameters	Dose (mg a.i./kg bw/day)
	0	100	300	1000
Number of mated females	24	24	24	24
Pre-terminal death or euthanasia	0	0	0	1
Number of non-pregnant females	0	1	0	0
Number of females with ≤ 5 implantation sites	0	0	0	0
Number of evaluated females on GD20 (Caesarean section)	24	23	24	23

Table 4: Summary of the intra-uterine evaluation

Parameter	Dose (mg a.i./kg bw/day)
	0	100	300	1000
Number of evaluated dams	24	23	24	23
Mean number of corpora lutea	12.08	11.75	11.75	12.04
Pre-implantation loss, mean	1.21	1.74	1.04	1.35
Pre-implantation loss (%), mean	9.19	13.93	8.64	10.26
Mean number of implantations	10.88	10.52	10.71	10.70
Early embryonic loss, mean	0.50	0.43	0.38	0.65
Early embryonic loss (%), mean	4.53	3.55	3.25	5.93
Late embryonic loss, mean	0.08	0.09	0.17	0.04
Late embryonic loss (%), mean	0.67	0.87	1.60	0.33
Dead foetuses, mean	0.00	0.00	0.00	0.00
Dead foetuses (%), mean	0.00	0.00	0.00	0.00
Post-implantation loss, mean	0.52	0.58	0.54	0.70
Post-implantation loss (%), mean	5.19	4.42	4.85	6.26
Total intra-uterine mortality, mean	1.79	2.26	1.58	2.04
Total intra-uterine mortality (%), mean	13.86	17.83	13.06	15.91
Viable foetuses, mean	10.29	10.00	10.17	10.00

Table 5: Examination of viable foetuses

Parameter	Dose (mg a.i./kg bw/day)
	0	100	300	1000
Number of examined litters	24	23	24	23
Viable foetuses, mean	10.19	10.00	10.27	10.00
Male foetuses, mean	5.50	4.91	4.83	4.70
Female foetuses, mean	4.70	5.09	5.33	5.30
Total number of foetuses	247	230	244	230
Total number of male foetuses	132	113	116	108
Total number of female foetuses	115	117	128	122
Sex distribution (% of males / females)	53/47	49/51	48/52	47/53
Mean foetal weight / litter (g)	3.449	3.457	3.453	3.390
Number of foetuses with retarded body weight	7	10	7	11
Number of affected litters (with runts)	6	4	6	7

Table 6: Summary table of the external abnormalities

Parameter	Dose (mg a.i./kg bw/day)
	0	100	300	1000
Total number of examined litters	24	23	24	23
Total number of examined foetuses	247	230	234	230
Total number of intact (normal) foetuses	230	211	220	213
Total number of foetuses / litters with malformation	0/0	0/0	0/0	0/0
Total number of foetuses / litters with variation	0/0	0/0	0/0	0/0

Notes: Numbers represent the number of abnormalities / number of affected litters.

Table 7: Summary table of the visceral abnormalities

Parameter	Dose (mg a.i./kg bw/day)
	0	100	300	1000
Total number of examined litters	24	23	24	23
Total number of examined foetuses	124	115	121	116
Total number of intact (normal) foetuses	118	110	116	112
Total number of foetuses/litters with malformations	2/2	0/0	2/2	3/3
Total number of foetuses/litters with variation	4/4	5/5	3/3	1/1

Notes: Numbers represent the number of abnormalities / number of affected litters.

Table 9: Summary table of the skeletal abnormalities

Parameter	Dose (mg a.i./kg bw/day)
	0	100	300	1000
Total number of examined litters	24	23	24	23
Total number of examined foetuses	123	115	123	114
Total number of intact (normal) foetuses	112	101	114	101
Total number of foetuses/litters with malformations	5/5	4/3	0/0	1/1
Total number of foetuses/litters with variation	6/5	10/8	9/7	12/7

Executive summary:

A developmental toxicity study (OECD TG 414) was performed to assess the effects of the test item on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (distilled water) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20.

The dose levels were set based on the available data and information from previous experimental work, including the results of an Oral (gavage) Dose Range Finding Toxicity Study in Pregnant Hannover Wistar Rats. Based on the results from the Dose Range Finding study, doses of 1000, 300 and 100 mg a.i./kg bw/day were selected for the main study (designated High, Mid and Low dose respectively). The aim was to use the highest dose of 1000 mg a.i./kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest

dose level.

Test item formulations were analysed for concentration twice during the treatment period using a validated HPLC-MS method. Simultaneously, vehicle control formulations were also analysed for the test item.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

The number of confirmed pregnant, evaluated dams was 24 in the Control and in the Mid dose group, and 23 in the Low and in the High dose groups.

Results

All test item formulations were within the range of 93.4-107.3% of nominal concentration, were stable (at least 10 days at 20°C) and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

One High dose female (4521) was found dead on Day 10. Decreased activity, piloerection, hunched back, tremor, red left eye/nose were clinically recorded for this female. The death was considered to be the result of a local effect of the test item.

Thin fur was observed in 1 out of 23 animals in the High dose group.

Piloerection was present in 1 out of 24 animals in the Mid group, and 3 out of 23 animals in the High dose group. It was apparent from Day 9 until Day 16, and occurred for up to six days.

Laboured respiration was observed in 1 out of 23 animals in the High dose group. Slight noisy respiration was present in 4 out of 23 animals in the High dose group. The finding appeared from Day 8 until Day 19. Noisy respiration can be a common local effect with surfactants. This may result from exceedingly small amounts entering the trachea (e.g. from reflux, after oral dosing) causing local irritation. Occasional deaths, due to reflux or similar respiratory exposure, after oral dosing with surfactants may be anticipated.

No test item related body weight decrease was observed in any of the dose groups.

A transient decrease of food consumption in the High dose group was observed at the start of the treatment.

Dark/red discoloration, of the non-collapsed lungs, seen in the female found dead (4521) may have been treatment related although this is a common agonal/post mortem incidental change. Enlarged adrenal glands were also observed macroscopically in this animal. There was no evidence for overt systemic toxicity in this animal.

No test item related macroscopic or microscopic findings were present at necropsy in the surviving females.

There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the controls.

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.

There was no difference in the number of runts between the control and treated groups.

There were no test item related effects on the external or visceral development of the foetuses in this study. The number of skeletal variations was higher in the test item treated groups when compared to the controls. Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and constituted the common range of background changes. They were, therefore, not regarded as a test item related.

In conclusion, the test item, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD 6 to GD 19 at 1000 mg a.i./kg bw/day induced no maternal systemic toxicity with no effects on body weight or growth. There was some evidence for local respiratory irritation at the High dose, which is considered as a common finding with orally dosed surfactants. No embryotoxicity or foetotoxicity was observed in this study following developmental and foetal examinations. There were no malformations considered attributable to the test item at any dose level.

The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity: 1000 mg a.i./kg bw/day

Based on no maternal systemic effects at 1000 mg a.i./kg bw/day.

NOAELembryotoxicity: 1000 mg a.i./kg bw/day

Based on the lack of any test-item related intra-uterine effect in any treatment group.

NOAELfoetotoxicity: 1000 mg a.i./kg bw/day

Based on no effect on runts at 1000 mg a.i./kg bw/day.

NOAELteratogenecity: 1000 mg a.i./kg bw/day

Based on the lack of treatment related malformations in any dose group.

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

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 developmental toxicity study (OECD TG 414) was performed to assess the effects of the test item on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (distilled water) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20.

The dose levels were set based on the available data and information from previous experimental work, including the results of an Oral (gavage) Dose Range Finding Toxicity Study in Pregnant Hannover Wistar Rats. Based on the results from the Dose Range Finding study, doses of 1000, 300 and 100 mg a.i./kg bw/day were selected for the main study (designated High, Mid and Low dose respectively). The aim was to use the highest dose of 1000 mg a.i./kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest dose level.

Test item formulations were analysed for concentration twice during the treatment period using a validated HPLC-MS method. Simultaneously, vehicle control formulations were also analysed for the test item.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

The number of confirmed pregnant, evaluated dams was 24 in the Control and in the Mid dose group, and 23 in the Low and in the High dose groups.

Results

All test item formulations were within the range of 93.4-107.3% of nominal concentration, were stable (at least 10 days at 20°C) and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

One High dose female (4521) was found dead on Day 10. Decreased activity, piloerection, hunched back, tremor, red left eye/nose were clinically recorded for this female. The death was considered to be the result of a local effect of the test item.

Thin fur was observed in 1 out of 23 animals in the High dose group.

Piloerection was present in 1 out of 24 animals in the Mid group, and 3 out of 23 animals in the High dose group. It was apparent from Day 9 until Day 16, and occurred for up to six days.

Laboured respiration was observed in 1 out of 23 animals in the High dose group. Slight noisy respiration was present in 4 out of 23 animals in the High dose group. The finding appeared from Day 8 until Day 19. Noisy respiration can be a common local effect with surfactants. This may result from exceedingly small amounts entering the trachea (e.g. from reflux, after oral dosing) causing local irritation. Occasional deaths, due to reflux or similar respiratory exposure, after oral dosing with surfactants may be anticipated.

No test item related body weight decrease was observed in any of the dose groups.

A transient decrease of food consumption in the High dose group was observed at the start of the treatment.

Dark/red discoloration, of the non-collapsed lungs, seen in the female found dead (4521) may have been treatment related although this is a common agonal/post mortem incidental change. Enlarged adrenal glands were also observed macroscopically in this animal. There was no evidence for overt systemic toxicity in this animal.

No test item related macroscopic or microscopic findings were present at necropsy in the surviving females.

There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the controls.

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.

There was no difference in the number of runts between the control and treated groups.

There were no test item related effects on the external or visceral development of the foetuses in this study. The number of skeletal variations was higher in the test item treated groups when compared to the controls. Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and constituted the common range of background changes. They were, therefore, not regarded as a test item related.

In conclusion, the test item, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD 6 to GD 19 at 1000 mg a.i./kg bw/day induced no maternal systemic toxicity with no effects on body weight or growth. There was some evidence for local respiratory irritation at the High dose, which is considered as a common finding with orally dosed surfactants. No embryotoxicity or foetotoxicity was observed in this study following developmental and foetal examinations. There were no malformations considered attributable to the test item at any dose level.

The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity: 1000 mg a.i./kg bw/day, based on no maternal systemic effects at 1000 mg a.i./kg bw/day.

NOAELembryotoxicity: 1000 mg a.i./kg bw/day, based on the lack of any test-item related intra-uterine effect in any treatment group.

NOAELfoetotoxicity: 1000 mg a.i./kg bw/day, based on no effect on runts at 1000 mg a.i./kg bw/day.

NOAELteratogenicity: 1000 mg a.i./kg bw/day, based on the lack of treatment related malformations in any dose group.

Justification for classification or non-classification

Based on the available data for the registered substance, classification for reproductive toxicity is not required.