Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/08/2020 - 12/11/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25th June 201

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 4-(hydrogen phosphonatomethyl)-2-oxo-1,4,2λ⁵-oxazaphosphinan-2-olate hydrogen {[(hydrogen phosphonatomethyl)(2-hydroxyethyl)amino]methyl}phosphonate

EC Number:

939-513-8

Molecular formula:

C4H12NNaO7P2, C4H11NNa2O7P2 and C4H10NNa3O7P2

IUPAC Name:

tetrasodium 4-(hydrogen phosphonatomethyl)-2-oxo-1,4,2λ⁵-oxazaphosphinan-2-olate hydrogen {[(hydrogen phosphonatomethyl)(2-hydroxyethyl)amino]methyl}phosphonate

Specific details on test material used for the study:

The dose levels of the test item were recalculated on 100 % of active ingredient HEBMP-H (Total Active Acid).

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Age at study initiation: 12 weeks old
- Weight at study initiation:
- Fasting period before study: not specified
- Housing: Before mating, 2 rats of the same sex were housed together in one cage; during mating period – one male and two females were housed together in one cage; pregnant females were housed individually during gestation.
- Diet (e.g. ad libitum): Complete pelleted diet for rats and mice, ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 – 70 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua pro iniectione

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose levels of the test item HEBMP (1-3Na) were recalculated on 100 % of active ingredient HEBMP-H (Total Active Acid) for the main study. The application forms (the test item in Aqua pro iniectione) were prepared daily just before administration. The calculated volumes of the test item and vehicle were measured into a glass beaker. The application form was stirred by magnetic stirrer (250 rpm) for 30 minutes and then during the administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua pro iniectione. Due to it being an ionised substance, additional water was used as a vehicle.
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 1mL per 100 g of body weight
- Lot/batch no. (if required): 2004010227

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the application form was checked by determination of a concentration P of the test item in three places of application form (at the bottom, in the middle and on the surface).
Stability of the application form was checked by analyses of the application form within 120 min (at the time 0, 30, 60, 90 and 120 min). Time interval 0 min represents for concentration 18.3 mL /100 mL the time after 30 minutes of mixing by magnetic stirrer (250 rpm) and for concentration 1.8 mL/100 mL the time after 30 minutes of mixing by magnetic stirrer (250 rpm).
the solution of the test item in vehicle at defined laboratory conditions is homogenous and stable at least for 120 minutes from a finalization of the application form preparation.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male : 2 females
- Length of cohabitation: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

from gestation day (GD) 5 to 19

Frequency of treatment:

Daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low Dose (LD)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Middle Dose (MD)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High Dose (HD)

No. of animals per sex per dose:

24 pregnant females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the oral (gavage) combined repeated dose toxicity study with reproduction/developmental toxicity screening test in rat, conducted according to OECD Test Guideline 422 and in compliance with GLP (Harlan Laboratories, 2013a). The doses were also selected in order to align with the doses tested in the 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP.
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were examined for vitality or mortality changes daily during the acclimatization, mating and pregnancy. The health condition was controlled daily during the acclimatization period, during the mating period and during pregnancy. During the administration period clinical observation was made in order to record possible clinical effects of the test item application and all changes in behaviour of females. Females were observed in natural conditions in their cages after application, once a day at the similar time each day.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: First weighing was performed on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy. Weight increments were computed as a mean per group (in grams).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was determined at three-day intervals; it coincided with the terms of body weight recording.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: During macroscopy, all orifices, the cranial, thoracic and abdominal cavities were examined. During macroscopic examination the thyroid glands were removed from all pregnant females and were preserved in fixation medium. The thyroid gland of control and high dose group females were examined at histopathology.

OTHER:
THYROID HORMONES: Yes
- Time schedule for examinations: The blood samples for this examination were taken from orbital plexus by glass micropipette under the light ether narcosis on the 20th day of pregnancy.
- Parameters: triiodothyronine (T3); thyroxine (T4); thyroid stimulating hormone (TSH).

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [one half per litter]
- Skeletal examinations: Yes: [second half per litter]
- Head examinations: Yes (as part of skeletal examinations): [second half per litter]
- Anogenital distance: Yes
- Individual body weight: Yes
- Sex: Yes

Statistics:

For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups. The results statistically significant on probability level 0.05 are indicated in the summary tables.

The parametric tests were used for statistical evaluation of:
• body weight of females
• corrected body weight (subtraction weight of uterus from surgery body weight of females)
• food consumption (per interval)
• mean weight of foetuses
• anogenital distance
• thyroid hormones
• biometry of thyroid gland (absolute and relative weight)
• biometry of uterus (absolute and relative weight)
• preimplantation (IUDE) and postimplantation (IUDL) losses

As the first step the test for normality (Shapiro-Wilk test) was performed. If the data were not normally distributed the transformation of data was performed (Box-Cox transformation). If the data were not normal distributed after transformation the non-parametric tests (Kruskal-Wallis Test and Mann-Whitney test) for comparison of the medians were performed.
If data were normally distributed after transformation, the Variance check (Levene’s test) to verify standard deviations within each group was used. One-Way ANOVA (probability level 0.05) was used to detect whether there were any significant differences amongst the means and then the post hoc statistical testing (Fisher's least significant difference - LSD test) for only statistical significant differences was performed.

The non-parametric tests were used for statistical evaluation of following parameters:
• number of corpora lutea, number of implantations, number of resorptions
• number of live foetuses (males, females, both sex)
• number of dead foetuses
The two-groups Mann-Whitney test (probability level 0.05) was applied.

The categorical data (skeletal foetal findings) were analyzed using the generalized linear mixed models with Poisson distribution.

Indices:

Preimplantation loss: Preimplantation loss = [(corpora lutea - implantations) / corpora lutea] x 100
Postimplantation loss: Postimplantation loss = (resorptions/implantations) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical changes indicating dysfunction of the organism were found in the control and treated groups during the whole study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No unscheduled death of maternal animals was recorded during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weights of treated maternal animals in all dose groups were comparable to the control group during whole study. No statistically significant changes were observed. The body weight increment was slightly decreased in the 350 mg/kg bw/day group and increased in the 100 mg/kg bw/day group in comparison with the control group. In the highest dose group body weight increment was comparable with the control group.
Corrected body weight of all treated females (the necropsy body weight of female minus weight of uterus) was not statistically significantly changed compared to the control females. Mean values of corrected body weight of treated females were similar to the control females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The average food consumption of all treated groups was comparable with the control group during the whole study. Statistically significant differences were not detected.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Uterus: The mean absolute and relative weights of the uterus were slightly increased in the 100 mg/kg bw/day group in comparison with control group. Statistically significant differences in uterus biometry were not detected in females of any group.
Thyroid gland: Absolute and relative weights of the thyroid glands were similar in the treated and control females. Statistically significant differences of thyroid weights were not detected in females of any dose level

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The uterus dilatation (the change probably related with oestrous cycle) was detected sporadically at the dose levels of 100 and 350 mg/kg bw/day. No finding related with treatment was noted at necropsy in treated females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid gland: Histological examination of thyroid glands revealed a spontaneous finding; a squamous cell cyst in one female in the 1000 mg/kg bw/day group. Because treatment-related changes were not recorded for the high dose group, histopathology of thyroid gland at doses 100 and 350 mg/kg bw/day was not performed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant differences were recorded in serum levels of thyroid hormones T3, T4 and TSH in females from treated groups against control females.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of implantations in treated groups were comparable with the control group. Preimplantation losses (IUDE) in all treated groups were similar or lower in comparison with control group. Postimplantation losses (IUDL) were increased in the 350 mg/kg bw/day group in comparison with control. Statistically significant differences were not detected in these all parameters.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of resorptions in treated groups were comparable with the control group.

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One dead foetus was recorded in the control group and two dead fetuses in the 100 mg/kg bw/day group. No dead foetuses were recorded for the mid and high dose groups. The lowest number of foetuses was recorded for the 350 mg/kg bw/day group, because the lowest number of pregnant females was in this group. However, the average total number of foetuses per litter was comparable across the treated groups and control group.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of non-pregnant females was 4, 4, 8 and 4, respectively for the four groups, 0, 100, 350 and 1000 mg/kg bw/day. The highest number of non-pregnant females was recorded for the dose of 350 mg/kg bw/day. Sperms were present in the vaginal smears of the non-pregnant females; however, no implantations or resorptions were recorded after necropsy. The females were concluded not to have been fertilized/pregnant. The administration of the test item started from the 5th day of pregnancy. Therefore, the non-pregnant status of the females was not related to the test item treatment.

Other effects:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea in treated groups were comparable with the control group.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks:

active acid

Basis for effect level:

other: No adverse effects observed in maternal animals.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of foetuses was slightly decreased in the 350 mg/kg bw/day group compared to the control group, without statistical and toxicological significance. Male foetuses were heavier than females in all groups.

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:

effects observed, non-treatment-related

Description (incidence and severity):

One dead foetus was found in the control group and two in the the 100 mg/kg bw/day goup. The dead foetuses could not be examined due to autolysis. Foetuses without a tail were recorded sporadically for doses of 100 and 1000 mg/kg bw/day. These findings were not treatment related and were of spontaneous origin. No other external changes were recorded.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Examination of foetal skeleton indicated mainly delayed development of the skeleton at all dose levels as well as in the control group.
Statistical evaluation was performed for 'number of foetuses with skeletal findings‘ and also for 'number of litters with skeletal findings‘.
Incomplete ossification of foetal cranium was observed during examination in all groups including the control group. The delayed development in parietal bone, interparietal bone, supraoccipital bone was not related to the treatment with because the occurence of these findings in litters was high in all groups, including the control group. There was a statistically significantly increased number of foetuses with incomplete ossification of supraoccipital bone in the mid dose group, but in the case of conversion to litter no statistical significance was detected. The proportions of litters with incomplete ossification of the squamosal part of temporal bone were similar or lower in treated groups compared to the control group. The proportions of litters with incomplete ossification of nasal and frontal bones were statistically significantly increased in the mid dose in comparison with control litters. This delayed development could be associated with the lower foetal body weight that was recorded for the mid dose group. The proportions of litters with incomplete ossification of arcus zygomaticus (15.00 % – 25.00 % – 33.33 % – 11.11 %) was increased in the mid dose group, but without statistical significance and dose dependence. Litters with holes in the supraoccipital bone were also observed in high percentage in all treated groups as well as in the control group.

Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded in foetuses of all treated groups as well as the control group. It is normal biological variability for ossification of sternebra to be incomplete on the 20th day of pregnancy. These findings were not related to the treatment, because the occurence of these findings was also high in the control litters and dose dependence for these findings was not recorded. Bipartite ossification of ossification sites of sternebra was detected sporadically.

Examination of vertebrae revealed dumbbell and bipartite ossification of vertebrae thoracic centrum in all test groups including the control group. The proportion of litters with bipartite ossification of vertebrae thoracic centrum was increased (close to statistical significance: Pvalue = 0.056) in the 1000 mg/kg bw/day group in comparison with control group (10.00 % – 25.00 % – 20.00 % – 50.00 %). This finding was also quantitatively compared with findings in the control group of other developmental study performed simultaneously at our facility. In this simultaneous study the incidence of bipartite ossification of vertebrae thoracic centrum was 21.05 % in control litters, which is also lower than the incidence in the high dose group (50.00 %) for the current study. This delayed vertebrae development is not related to the lower body weight of foetuses. The foetal body weight at 1000 mg/kg bw/day was comparable with foetal body weight in the control group. The bipartite ossification of vertebrae thoracic centrum is included in the Transitional Findings (grey zone). These may be upgraded to malformation or downgraded to variations status, depending on severity and/or frequency of occurrence. Ossification delay is a transient finding that may indicate a delayed schedule of events but does not indicate disrupted development. Changes in the size, shape, or symmetry of sternebrae or vertebral centra are transient and have no implications for the health or survival of the offspring .

Asymmetric ossification of vertebrae thoracic centrum was recorded sporadically in foetuses. The proportions of litters with dumbbell ossification of vertebrae thoracic centrum were similar or lower in treated groups than in the control group. Test item treatment relation was not evident for these findings associated with vertebrae.

Changes such as wavy ribs and ribs-supernumerary site were detected also in all groups. The proportion of litters with wavy ribs was similar or lower in treated groups than in the control group. These findings did not relate to treatment with the test item. The proportion of litters with ribs-supernumerary site was increased in the high dose group in comparison with controls (35.00 % – 35.00 % – 26.67 % – 50.00 %), this increase is not dose-dependent and moreover statistical evaluation revealed that it is non-significant. This finding was also quantitatively comparable with findings in the control group of other developmental study performed simultaneously at the test facility. In this simultaneous study the incidence of ribs-supernumerary site was 57.89 % in control litters, which is comparable with the incidence in the high dose group (50.00 %) recorded in the current study. This change is not considered to be adverse.

Incomplete ossification of scapula was recorded only in treated groups. The proportions of litters with scapula was 0.00 % – 15.00 % – 20.00 % – 11.11 %, respectively for the four groups. However, this finding was recorded in a relatively low number of foetuses in the treated groups (0 – 3 – 3 – 2). The occurence of this finding was sporadic. Test item treatment relation was not evident for this sporadic variation.

Visceral malformations:

no effects observed

Description (incidence and severity):

No adverse findings were observed in any of the test groups or the control group.

Other effects:

no effects observed

Description (incidence and severity):

The AGD and corrected AGD of male and female foetuses at all doses were comparable with control group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: scapule

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

- cranial bones: delayed development at all dose groups and the control, non-treatment related
- sternebra: delayed development at all dose groups and the control, non-treatment related
- vertebrae: dumbbell and bipartite ossification of vertebrae thoracic centrum all dose groups and the control, non-treatment related
- ribs: wavy ribs and ribs-supernumerary site in all groups, non-statistically significant compared to control, non-treatment related
- scapula: incomplete ossification in all dose groups with evidence for treatment relevance

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

TNotes for tables 20 - 23:          

i.o.      – incomplete ossification

u.        – unossified

d.o.     – dumbbell ossification

b.o.     – bipartite ossification

as.os.  – asymmetric ossification

able 1: Pregnancy results

Group code	Number of pregnant females	Number of pregnant females	Number of Non-pregnant females**
	Number of females with foetuses	Number of females without live foetuses but with implantations and resorptions**
0	20	0	4 (102,108,116, 122)
100	20	0	4 (127,128,130,134)
350	15	1 (164)	8 (152,154,155,156,157,158,163,169)
1000	20	0	4 (180,183,184,197)

Note:  **numbers in parentheses = individual labels of single animals.

Only the data from females with live foetuses were used for calculations of means. Data from females with uterus implantations were used for calculation of preimplantation (IUDE) and postimplantation (IUDL) losses.

Table 2: Body weight in grams (mean ± standard deviation) 

Day of pregnancy	Group	Group	Group	Group
	0	100	350	1000
1^stday	277.7± 14.9	277.8± 14.1	276.4 ± 17.1	273.8 ± 11.9
5^thday	296.9 ± 18.5	294.4± 16.4	291.6 ± 17.0	294.4 ± 14.4
8^thday	305.5 ± 16.8	304.9 ± 16.9	300.8 ± 18.8	305.6 ± 14.1
11^thday	322.8 ± 20.1	324.1 ± 19.6	316.6 ± 21.5	322.1 ± 16.3
14^thday	339.2 ± 19.1	342.0 ± 18.0	332.2 ± 23.1	340.6 ± 18.6
17^thday	376.5 ± 29.3	380.2 ± 21.6	365.4 ± 33.7	375.4 ± 25.9
20^thday	423.1 ± 40.1	433.8 ± 28.2	414.5 ± 41.8	419.3 ± 31.5
Mean increment	145.4	156.1	138.0	145.5

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 3: Food consumption /animal/day (grams)

Day of pregnancy	Group	Group	Group	Group
	0	100	350	1000
5^thday	24.01	23.22	21.87	22.48
8^thday	26.73	26.67	24.94	25.02
11^thday	26.75	28.28	26.63	27.00
14^thday	29.19	29.63	28.45	28.93
17^thday	29.22	30.38	28.54	28.69
20^thday	32.00	34.26	31.95	31.72

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 4: Clinical observation

Week of pregnancy	Group	Group	Group	Group
	0	100	350	1000
1^stweek	1	1	1	1
2^ndweek	1	1	1	1
3^rdweek	1	1	1	1

Note:1–no clinical signs of intoxication

Table 5: Biometry of uterus (mean ± standard deviation)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Mean necropsy body weight of females (g)	423.1 ± 40.1	433.8 ± 28.2	414.5 ± 41.8	419.3 ± 31.5
Mean absolute weight of uterus (g)	79.94 ± 27.21	90.54 ± 15.27	79.45 ± 26.61	78.09± 24.13
Mean relative weight of uterus (%)	18.56 ± 5.39	20.89 ± 3.38	18.81 ± 5.28	18.35 ± 5.08

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 6: Body weight - corrected* (mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Body weight(g)	343.2 ± 23.5	343.3 ± 27.9	335.0 ± 23.5	341.2 ± 17.4

Note:*body weight correction = necropsy body weight of female – weight of uterus

Statistically significant differences on probability level 0.05 were not detected.

Table 7: Macroscopic findings (number of females with pathological findings)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Number of examined females	24	24	24	24
Number of died females	0	0	0	0
Without pathological findings	24	24	24	24
Uterus: dilatation	0	1	1	0

Note:Uterus dilatation = probablynon-pathological finding.

Table 8: Parameters of reproduction (number per female, mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
implantations	14.55± 4.22	16.65± 1.76	14.19± 5.87	14.70± 4.68
resorptions	0.70± 1.03	1.00± 2.10	0.75± 1.24	0.60± 0.88
corpora lutea	16.35± 2.32	17.45± 1.39	15.19± 5.00	15.75± 3.77

Table 9: IUDE and IUDL (% per female, mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
IUDE	12.33± 20.42	4.61± 6.23	9.46± 16.96	10.01± 18.00
IUDL	5.71± 8.93	5.48± 10.86	9.23± 24.75	4.34± 7.05

Note: Statistically significant differences on probability level 0.05 were not detected.

The mean of preimplantation and postimplantation losses were calculated from individual data of females.

IUDE = Preimplantation losses

IUDL = Postimplantation losses

Table 10: Weight of thyroid gland (group mean ± SD)

Thyroid gland	Group	Group	Group	Group
	0	100	350	1000
Absolute weight (g)	0.0311 ± 0.0017	0.0305 ± 0.0012	0.0306 ± 0.0011	0.0308 ± 0.0013
Relative weight (%)	0.0074 ± 0.0008	0.0071 ± 0.0005	0.0075 ± 0.0008	0.0074 ± 0.0006

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 11: Pregnant females - Thyroid hormones (mean concentration ± SD)

Hormone	Group	Group	Group	Group
	0	100	350	1000
T3(ng/mL)	0.648 ± 0.056	0.652 ± 0.068	0.674 ± 0.085	0.623 ± 0.062
T4(µg%)	3.368 ± 0.525	3.271 ± 0.558	3.230 ± 0.428	3.086 ± 0.434
TSH(pg/mL)	809.5 ± 216.0	869.6 ± 223.5	776.2 ± 180.9	812.0 ± 225.0

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 12: Number of foetuses (total in group)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Total number of live foetuses	277	313	215	282
Number of live foetuses – males	142	162	112	141
Number of live foetuses – females	135	151	103	141
Number of dead foetuses	1	2	0	0

Table 13: Number of foetuses (average per litter; mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
Total number of live foetuses	13.85 ± 4.56	15.65 ± 1.95	14.33 ± 5.25	14.10 ± 4.73
Number of live foetuses – males	7.10 ± 2.71	8.10 ± 1.97	7.47 ± 3.29	7.05 ± 2.61
Number of live foetuses – females	6.75 ± 3.34	7.55 ± 1.32	6.87 ± 2.88	7.05 ± 2.95
Number of dead foetuses	0.05 ± 0.22	0.10 ± 0.31	0.00 ± 0.00	0.00 ± 0.00

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 14: Body weight of foetuses (grams, mean ± SD)

Parameter	Group	Group	Group	Group
	0	100	350	1000
weight of all foetues	3.80± 0.73	3.80± 0.74	3.57± 0.56	3.77± 1.12
weight of male foetus	3.89± 0.72	3.91± 0.76	3.68 ± 0.64	3.86± 1.16
weight of female foetus	3.70± 0.73	3.68± 0.74	3.46 ± 0.54	3.56± 0.99

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 15: Mean anogenital distance of foetuses (mm)

Group code	0	0	100	100	350	350	1000
Sex	Male	Female	Male	Female	Male	Female	Male	Female
Mean AGD	3.57	2.18	3.60	2.18	3.56	2.18	3.39	2.17
Corrected AGD	2.28	1.42	2.30	1.42	2.31	1.44	2.18	1.45

Note: Statistically significant differences on probability level 0.05 were not detected.

Table 16: External alterations - foetuses

Alteration	Group	Group	Group	Group
	0	100	350	1000
Total number of examined foetuses	277	313	215	282
Total number of examined litters	20	20	15	20
Dead foetus	1	2	0	0
Foetus without tail	0	1	0	1
Number of examined foetuses in litter (mean ± SD)	13.85 ±4.56	15.65 ±1.95	14.33 ±5.25	14.10 ±4.73
Total number of foetuses with alteration	1	3	0	1
Number of foetuses with alteration in litter(mean ± SD)	0.05 ±0.22	0.15 ±0.49	0.00 ±0.00	0.05 ±0.22
Proportion of foetuses with alteration in litter (% mean ± SD)	0.36 ±1.60	0.88 ±2.88	0.00 ±0.00	0.31 ±1.40

Table 17: Macroscopic changes of soft tissues – individual foetuses

Alteration	Group	Group	Group	Group
	0	100	350	1000
Total number of examined foetuses	129	147	100	130
Total number of examined litters	20	20	15	20
With pathological findings - total(number of affected foetuses)	0	0	0	0
Number of examined foetuses in litter (mean ± SD)	6.45 ±2.31	7.35 ±0.99	6.67 ±2.55	6.50 ±2.35
Total number of foetuses with alteration	0	0	0	0
Number of foetuses with alteration in litter (mean ± SD)	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00
Proportion of foetuses with alteration in litter (% mean ± SD)	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00

Table 18: Skeletal alterations (number of affected foetuses)

Group code	0	100	350	1000
Number of examined foetuses	148	165	112	128
CRANIUM
Nasal bone – i.o.	4	0	24	7
Frontal bone – i.o.	7	6	17	7
Parietal bone – i.o.	45	59	37	29
Interparietal bone–i.o.	64	92	69	56
Supraoccipital bone – i.o.	97	115	93	80
Supraoccipital bone – hole	29	28	18	38
Arcus zygomaticus–i.o.	4	7	8	4
Squamosal part of temporal bone – i.o.	12	18	15	8
Basisphenoid–i.o.	1	1	0	0
STERNEBRA
Sternebra – ossification sites – i.o.	137	144	106	120
Sternebra – ossification sites – u.	73	65	63	53
Sternebra – ossification sites – b.o.	0	0	0	2
VERTEBRAE
Vertebrae thoracic centrum – b.o.	2	11	3	14
Vertebrae thoracic centrum – b.o., as.os.	0	0	2	2
Vertebrae thoracic centrum – d.o.	36	58	33	59
RIBS
Ribs – supernumerary site	23	15	11	16
Ribs – wavy	8	9	2	3
Scapula – i.o.	0	3	3	2

Note:The resultsstatistically significantly changedon probability level 0.05 were shaded in the summary table

Table 19: Skeletal alterations (number of affected litters)

Group code	0	100	350	1000
Number of examined litters	20	20	15	18
CRANIUM
Nasal bone – i.o.	3	0	9	5
Frontal bone – i.o.	5	4	9	6
Parietal bone – i.o.	17	16	12	14
Interparietal bone–i.o.	17	19	14	16
Supraoccipital bone – i.o.	19	19	15	17
Supraoccipital bone – hole	15	16	10	13
Arcus zygomaticus–i.o.	3	5	5	2
Squamosal part of temporal bone – i.o.	7	6	6	5
Basisphenoid–i.o.	1	1	0	0
STERNEBRA
Sternebra – ossification sites – i.o.	20	20	15	18
Sternebra – ossification sites – u.	15	14	12	13
Sternebra – ossification sites – b.o.	0	0	0	2
VERTEBRAE
Vertebrae thoracic centrum – b.o.	2	5	3	9
Vertebrae thoracic centrum – b.o., as.os.	0	0	2	2
Vertebrae thoracic centrum – d.o.	15	17	10	16
RIBS
Ribs – supernumerary site	7	7	4	9
Ribs – wavy	6	7	1	3
Scapula – i.o.	0	3	3	2

Note:Statistically significant differences on probability level 0.05 were not detected.

Table 20: Skeletal alterations (% proportion of litters with affected foetuses)

Group code	0	100	350	1000
Number of examined litters	20	20	15	18
Alteration
CRANIUM
Nasal bone – i.o.	15.00	0.00	60.00	27.78
Frontal bone – i.o.	25.00	20.00	60.00	33.33
Parietal bone – i.o.	85.00	80.00	80.00	77.78
Interparietal bone–i.o.	85.00	95.00	93.33	88.89
Supraoccipital bone – i.o.	95.00	95.00	100.00	94.44
Supraoccipital bone – hole	75.00	80.00	66.67	72.22
Arcus zygomaticus–i.o.	15.00	25.00	33.33	11.11
Squamosal part of temporal bone – i.o.	35.00	30.00	40.00	27.78
Basisphenoid–i.o.	5.00	5.00	0.00	0.00
STERNEBRA
Sternebra – ossification sites – i.o.	100.00	100.00	100.00	100.00
Sternebra – ossification sites – u.	75.00	70.00	80.00	72.22
Sternebra – ossification sites – b.o.	0.00	0.00	0.00	11.11
VERTEBRAE
Vertebrae thoracic centrum – b.o.	10.00	25.00	20.00	50.00
Vertebrae thoracic centrum – b.o., as.os.	0.00	0.00	13.33	11.11
Vertebrae thoracic centrum – d.o.	75.00	85.00	66.67	88.89
RIBS
Ribs – supernumerary site	35.00	35.00	26.67	50.00
Ribs – wavy	30.00	35.00	6.67	16.67
Scapula – i.o.	0.00	15.00	20.00	11.11

Table 21: Skeletal alterations (% proportion of affected foetuses in litter (mean ± SD)

Group code	0	100	350	1000
Number of examined foetuses	148	165	112	128
Number of examined litters	20	20	15	18
Alteration
CRANIUM
Nasal bone – i.o.	2.22 ± 5.81	0.00 ± 0.00	20.42 ± 26.05	5.38 ± 10.68
Frontal bone – i.o.	3.96  ± 7.53	3.58 ± 7.84	14.58 ± 16.56	5.62 ± 10.28
Parietal bone – i.o.	30.54 ± 23.12	34.63 ± 28.39	29.03 ± 23.15	23.52 ± 22.55
Interparietal bone–i.o.	40.74  ± 29.42	55.08 ± 30.42	63.17 ± 32.26	44.91 ± 32.78
Supraoccipital bone – i.o.	63.44  ± 27.67	69.44 ± 27.24	81.02 ± 20.60	59.56 ± 30.89
Supraoccipital bone – hole	21.74 ± 20.49	17.39 ± 14.06	14.88 ± 15.67	30.39 ± 26.96
Arcus zygomaticus–i.o.	2.29  ± 5.93	3.85 ± 7.26	6.74 ± 11.35	2.64 ± 9.01
Squamosal part of temporal bone – i.o.	7.08 ± 11.70	10.13 ± 18.18	12.22 ± 17.39	5.93 ± 12.66
Basisphenoid–i.o.	0.56 ± 2.48	0.56 ± 2.48	0.00 ± 0.00	0.00 ± 0.00
STERNEBRA
Sternebra – ossification sites – i.o.	93.42  ± 7.93	86.50 ± 20.63	95.49 ± 5.79	94.24 ± 12.56
Sternebra – ossification sites – u.	49.54 ± 36.67	40.32 ± 32.27	48.06 ± 34.69	37.48 ± 29.86
Sternebra – ossification sites – b.o.	0.00  ± 00.00	0.00  ± 00.00	0.00 ± 0.00	1.17 ± 3.42
VERTEBRAE
Vertebrae thoracic centrum – b.o.	1.11  ± 3.42	6.74 ± 15.38	2.36 ± 4.96	8.70 ± 10.04
Vertebrae thoracic centrum – b.o., as.os.	0.00 ± 0.00	0.00 ± 0.00	1.62 ± 4.35	1.41 ± 4.14
Vertebrae thoracic centrum – d.o.	25.07  ± 21.52	34.66 ± 25.34	29.43 ± 28.30	41.83 ± 28.89
RIBS
Ribs – supernumerary site	13.61 ± 23.68	8.79 ± 15.00	10.14 ± 19.36	10.58 ± 13.17
Ribs – wavy	5.10 ± 9.59	5.26 ± 7.74	1.67 ± 6.45	3.10 ± 8.38
Scapula – i.o.	0.00 ± 0.00	1.89  ± 4.66	3.12 ± 6.62	1.35 ± 3.99

Notes for tables 18 - 21:          

i.o. – incomplete ossification

u. – unossified

d.o. dumbbell ossification

b.o. - bipartite ossification

as.os. – asymmetric ossification

See attachment for individual animal data.

Applicant's summary and conclusion

Conclusions:

In the pre-natal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal toxicity and developmental effects was concluded to be at least 1000 mg active acid/kg bw/day based on no treatment-related adverse effects in maternal animals and foetuses at any dose level.