Potassium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 K:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10.03.2020 – 26.10.2020

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 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 K:1)

EC Number:

701-184-1

Molecular formula:

HMDTMP-4K C10H24K4N2O12P4 HMDTMP-5K C10H23K5N2O12P4 HMDTMP-6K C10H22K6N2O12P4 HMDTMP-7K C10H21K7N2O12P4

IUPAC Name:

Potassium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 K:1)

Specific details on test material used for the study:

The dose levels of the test item Dequest 2054 (HMDTMP (4-7K)) were recalculated on 100 % of active ingredient HMDTMP-H (Total Active Acid) for the determination of the stability and homogeneity of the application form and main study.
The active ingredient of test item is the acid form, HMDTMP-H (Total Active Acid). The substance has been supplied as a 22.6% w/w Total Active Acid aqueous solution. The water can be considered as an additive. Due to this being an anionised substance, additional water should be used as vehicle. The density of HMDTMP (4-7K) solution is 1.28 g/ml. Therefore, a dose of 1000 mg/kg bw/day of the active ingredient is equivalent to approximately 3.5 ml HMDTMP (4-7K) solution/kg body weight/day.

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
- Weight at study initiation: not specified
- Fasting period before study: no
- Housing: SPF conditions according to internal SOP No.
Pre-mating period: 2 rats of the same sex in one cage,
Mating period: one male and two females in one cage
Gestation: Pregnant females were housed individually
- Diet (e.g. ad libitum): Complete pelleted diet for rats and mice in SPF breeding
- Water (e.g. ad libitum): Free access to drinking water, ad libitum
- Acclimation period: 6 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:

other: Aqua pro iniectione

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The application form of the test item, recalculated to 100 % w/w Total Active Acid HMDTMP-H. 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 (500 rpm) for 30 minutes and then during the administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua pro iniectione was chosen as vehicle because the substance is anionised and additional water should be used as vehicle.
- Concentration in vehicle: Not specified
- Amount of vehicle (if gavage): The concentrations of all dose levels of HMDTMP-H were adjusted to ensure the administration of 1 mL 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:

The stability and the homogeneity of application form were determined at the test facility by means of measuring the concentrations of K and P (mg/kg) in the test item application form.
Homogeneity of the application form was checked by determination of a concentrations K and P of the test item in three places of the 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).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 2 females
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

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:

vehicle only

Doses / concentrations 2

Dose / conc.:

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

Remarks:

active acid

Doses / concentrations 3

Dose / conc.:

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

Remarks:

active acid

Doses / concentrations 4

Dose / conc.:

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

Remarks:

active acid

No. of animals per sex per dose:

- total number of animals before mating: 100 females and 25 males. Males were not treated and were used only for mating purpose.
- during gestation: 24 probably pregnant females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on no adverse effects in a dose range-finding study in which pregnant female rats were given daily oral gavage doses of 250, 500 and 1000 mg active acid/kg bw/day throughout gestation.
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily - during the acclimatization, mating and pregnancy
- Cage side observations checked included: vitality or mortality changes, general health condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Females were observed in natural conditions in their cages after application, once a day at the similar time each day.

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: During macroscopy, all orifices, the cranial, thoracic and abdominal cavities were examined. The thyroid glands of control and high dose females were examined at histopathology.

OTHER:
THYROID HORMONE ANALYSIS:
- Time schedule for examinations: on GD 20 prior to termination
- Parameters examined: Blood samples from the pregnant females were assessed for serum levels of thyroid hormones (T3 - Triiodothyronine, T4 - Thyroxine, TSH- Thyroid Stimulating Hormone).

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: [second half per litter]
- Anogenital distance: Yes [all per litter]
- Sex: Yes [all per litter]
- Individual body weights: Yes [all per litter]

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 signs of toxicity were recorded during the treatment period in the control and treated females of all dose levels

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One high dose female died on the 5th day of pregnancy after first administration of the test substance. The cause of death was an intubation error (female No. 196).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weights of treated females at all dose levels were similar to the control group throughout the study. No statistically significant changes were observed. The body weight increments at all dose levels were comparable with the body weight increment of 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 throughout the 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:

no effects observed

Description (incidence and severity):

The mean absolute and relative weights of uterus at all dose levels were comparable with the control group. Statistically significant differences in uterus biometry were not detected for any of the treated females.
Absolute and relative thyroid gland weights were comparable between the treatment and control females. Statistically significant differences of thyroid weights were not detected.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Uterus dilatation was observed in 2 control group females and 3 middle dose (500 mg active acid/kg bw/day) females. This change is considered to be related to oestrous cycle and since no dose-relationship was observed it is concluded to be not a treatment-related adverse finding. Overall, there were no treatment-related gross pathological findings in any of the treatment group or control group females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histological examination of thyroid glands revealed no pathological changes. Because treatment related changes were not recorded for the high dose group, histopathology of thyroid glands at doses of 125 and 500 mg active acid/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 when compared to control females.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation losses (IUDE) and pos-timplantation losses (IUDL) were comparable between the treatment and the control groups. Statistically significant differences were not detected.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of resorptions were comparable between the treatment and the control groups. Statistically significant differences were not detected.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of resorptions were comparable between the treatment and the control groups. Statistically significant differences were not detected.

Dead fetuses:

no effects observed

Description (incidence and severity):

There was one dead foetus in the middle dose level (500 mg active acid/kg bw/day). Overall, the number of foetuses was comparable between all groups. 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 females with foetuses on the 20th day of pregnancy was comparable between all treated groups and the control group.

Other effects:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea were comparable between the treatment and the control groups. Statistically significant differences were not detected.

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 treatment-related adverse effects observed

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The foetal mean body weight was slightly decreased in the low dose group (125 mg active acid/kg bw/day) when 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):

The dead foetus at 500 mg active acid/kg bw/day group could not be examined for external malformations due to autolysis. At 500 mg active acid/kg bw/day group, there was one male foetus without a tail and with absent end part of the spine. Since these findings were observed in one foetus from one middle dose litter, they were not considered to be treatment-related and were concluded to be of spontaneous origin. No other external changes were recorded for any of the foetuses.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Incomplete ossification of foetal cranium was observed during the examination in all groups, including the control group. The delayed development in parietal bone, interparietal bone, supraoccipital bone, squamous part of temporal bone and arcus zygomaticus was not related to the treatment because the occurence of these findings in litters were high in all groups, including the control group. There were detected statistically significantly increased numbers of foetuses with incomplete ossification of parietal bone, interparietal bone, supraoccipital bone, arcus zygomaticus and squamosal part of temporal bone at 125 mg active acid/kg bw/day in comparison with control. The increased proportions of litters with incomplete ossification premaxilla, maxilla and frontal bone were also detected at the lowest dose level compared to the control, but without statistical significance. These findings in the lowest dose level were without toxicological significance. The litters with holes in the supraoccipital bone were also detected in high percentage in all treated groups as well as in the control group. Other changes of foetal cranium were found only sporadically.
Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded in foetuses of all treated groups as well as in the control group. It is a normal variability in the schedule of ossification, ossification of the sternum have not to be complete by the 20th day of gestation. These findings were not related to the treatment, because the occurence of these findings was high also in the control litters and dose dependence for these findings was not recorded.
Examination of vertebrae revealed dumbbell and bipartite ossification of vertebrae thoracic centrum in all test groups including control group. The proportions of litters with bipartite ossification of vertebrae thoracic centrum were comparable in treated groups with control group. The proportions of litters with dumbbell ossification of vertebrae thoracic centrum were slightly increased in treated groups in comparison with control group (68.42 % – 84.21 % – 73.68 % – 89.47 %), without statistical significance and dose dependence. The asymmetric ossification of vertebrae thoracic centrum was recorded sporadically in foetuses.The dumbbell ossification of vertebrae lumbar centrum was detected only in litters at treated groups (0 % – 15.79 % – 15.79 % – 15.79 %), but this finding was recorded in relatively low number of foetuses in the treated groups (0 – 4 – 3 – 3). The occurence of this finding was sporadic. Therefore, a dose-response relationship for the findings relating to the vertebrae was not evident.
The malformations, misshapen centrum of lumbar vertebrae and absent centrums of lumbar and sacral vertebrae, were recorded only in one foetus at the dose level 500 mg active acid/kg bw/day (in litter from female No.168 – male foetus No.1). These malformations observed in this one litter in one female were not treatment related and were probably of spontaneous origin.
The changes such as wavy ribs and ribs-supernumerary site were detected also in all groups, including the control group. The proportion of litters with ribs-supernumerary site was slightly increased in the mid dose group in comparison with the control (57.89 % – 63.16 % – 78.95 % – 63.16 %). The proportions of litters with wavy ribs were increased at all dose levels in comparison with the control group (5.26 % – 47.37 % – 21.05 % – 26.32 %), this increase is not related to the test item as statistical significance was not reached. Furthermore, wavy ribs are considered to be a transient finding in rats. This finding was also quantitatively comparable with findings in the control group of other developmental studies performed at the testing facility. Therefore, occurrence of this variation was not considered to be of adverse nature in this study.
The branched rib and the malformation of full supernumerary rib were recorded in one foetus at the 500 mg active acid/kg bw/day dose level (in litter from female No. 168 – female foetus No.6). These malformations observed in this one litter in one female were not treatment related and were probably of spontaneous origin.
Misshapen scapula was recorded only in one 500 mg active acid/kg bw/day group foetus (litter from female No.168 – female foetus No.5). The incomplete ossification of scapula was recorded at all groups. The proportion of litters with incomplete ossification of scapula was slightly increased at the 125 mg active acid/kg bw/day dose level in comparison with the control (47.37 % – 63.16 % – 42.11 % – 47.37 %), without toxicological significance.

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 anogenital distance (AGD) and corrected AGD of male and female foetuses at all dose levels were comparable with control group.

Effect levels (fetuses)

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 treatment-related adverse effects observed.

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: scapule

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

- foetal cranium - non-treatment related incomplete ossification at all dose groups and control group; statistically significantly increased number of foetuses with incomplete ossification of parietal bone, interparietal bone, supraoccipital bone, arcus zygomaticus and squamosal part of temporal bone only at 125 mg/kg bw/day, not considered treatment-related; non-statistically significant increased proportions of litters with incomplete ossification of premaxilla, maxilla and frontal bone at 125 mg/kg bw/day and control group.
- sternebra - non-treatment related incomplete ossification at all dose groups and control group.
- vertebrae - non-treatment related dumbbell and bipartite ossification of vertebrae thoracic centrum at all dose groups and control group; sporadic without dose-response relationship dumbbell ossification of vertebrae lumbar centrum at treated groups; non-treatment related and of spontaneour origin misshapen centrum of lumbar vertebrae and absent centrums of lumbar and sacral vertebrae in one 500 mg/kg bw/day foetus.
- ribs - wavy ribs at all dose groups and control group with non-statistically significant increase in the treatment groups when compared to controls; non-treatment related branched rib in one 500 mg/kg bw/day foetus.
- supernumerary ribs - ribs-supernumerary site at all dose groups and control group with non-statistically significant increase in the middle dose group when compared to controls; non-treatment related full supernumerary rib in one 500 mg/kg bw/day foetus.
- scapula - non-treatment related incomplete ossification of scapula at all dose groups and control group; misshapen scapula in one 500 mg/kg bw/day foetus.

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Pregnancy results

Group	Number of females with foetuses	Number of non-pregnant females**	Number of females without live foetuses but with implantations and resorptions**
0	22	2 (120, 121)	0
125	21	3 (143, 144, 148)	0
500	20	4 (157, 160, 169, 170)	0
1000*	20	3 (188, 192, 198)^*	0

Note:  *female No. 196 died on the 5^thday of the pregnancy – intubation error

           **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 code	Group code	Group code	Group code
	0	125	500	1000
1^stday	276.2± 13.6	281.3± 16.2	278.6 ± 14.5	281.3 ± 12.8
5^thday	294.1 ± 13.1	299.1± 14.7	292.3 ± 10.2	297.2 ± 12.2
8^thday	305.1 ± 14.5	309.2 ± 15.5	303.0 ± 13.1	307.8 ± 14.2
11^thday	322.4 ± 16.3	325.2 ± 16.6	318.3 ± 14.8	325.2 ± 14.5
14^thday	339.6 ± 16.2	341.9 ± 18.9	335.8 ± 15.6	341.8 ± 14.6
17^thday	375.8 ± 23.0	377.4 ± 20.0	372.3 ± 19.8	377.9 ± 18.9
20^thday	425.3 ± 29.0	431.3 ± 27.2	425.0 ± 25.1	427.6 ± 25.5
Mean increment	149.1	150.0	146.4	156.0

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

Table 3: Food consumption g/animal/day(mean)

Day of pregnancy	Group code	Group code	Group code	Group code
	0	125	500	1000
5^thday	22.78	23.93	23.06	23.37
8^thday	24.91	25.47	25.18	24.49
11^thday	26.24	27.20	25.99	25.64
14^thday	27.67	28.16	28.59	27.67
17^thday	29.12	27.85	29.18	29.01
20^thday	30.91	30.58	32.20	31.22

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

Table 4: Health condition control

Week of pregnancy	Group code	Group code	Group code	Group code
	0	125	500	1000
1^stweek	1	1	1	1
2^ndweek	1	1	1	1
3^rdweek	1	1	1	1

Note:  1– physiological appearance

Table 5: Clinical observation

Week of pregnancy	Group code	Group code	Group code	Group code
	0	125	500	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 6: Biometry of uterus(mean± standard deviation)

Parameter	Group code	Group code	Group code	Group code
	0	125	500	1000
Mean necropsy body weight of females (g)	425.3 ± 29.0	431.3 ± 27.2	425.0 ± 25.1	427.6 ± 25.5
Mean absolute weight of uterus (g)	85.56 ± 21.71	87.99 ± 13.85	87.71 ± 15.34	86.35± 11.27
Mean relative weight of uterus (%)	19.94 ± 4.42	20.37 ± 2.71	20.55 ± 2.99	20.15 ± 1.81

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

Table 7: Body weight - corrected* (mean ± SD)

Parameter	Group code	Group code	Group code	Group code
	0	125	500	1000
Body weight(g)	339.8 ± 18.5	343.3 ± 22.4	337.2 ± 16.7	341.2 ± 18.1

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

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

Table 8: Macroscopic findings(number of females with pathological findings)

Parameter	Group code	Group code	Group code	Group code
	0	125	500	1000
Number of examined females	24	24	24	24
Number of died females	0	0	0	1*
Without pathological findings	24	24	24	23
Uterus: dilatation	2	0	3	0

Note:  Uterus dilatation = probablynon-pathological finding

           *Female No. 196 – died on the 5^thday of the pregnancy – intubation error

Table 9: Parameters of reproduction(number per female, mean ± SD)

Parameter	Group code	Group code	Group code	Group code
	0	125	500	1000
implantations	15.32± 3.27	16.81± 1.81	16.00± 2.68	16.05± 1.39
resorptions	0.59± 1.14	0.52± 0.87	0.75± 1.68	0.50± 0.76
corpora lutea	16.05± 2.06	17.24± 1.81	16.65± 1.90	16.75± 1.45

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

Table 10: IUDE and IUDL (% per female, mean ± SD)

Parameter	Group Code	Group Code	Group Code	Group Code
	0	125	500	1000
IUDE	5.20± 14.63	2.44± 3.72	4.27± 9.72	4.05± 4.96
IUDL	3.65± 7.17	3.18± 5.24	4.43± 9.45	3.05± 4.65

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 11: Weight of thyroid gland (group mean±SD)

Thyroid gland	Group code	Group code	Group code	Group code
	0	125	500	1000
Absolute weight (g)	0.0312 ± 0.0015	0.0311 ± 0.0015	0.0308 ± 0.0014	0.0312 ± 0.0014
Relative weight (%)	0.0074 ± 0.0006	0.0072 ± 0.0006	0.0073 ± 0.0006	0.0073 ± 0.0005

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

Table 12: Pregnant females - Thyroid hormones (mean concentration ± SD)

Hormone	Group code	Group code	Group code	Group code
	0	125	500	1000
T3(ng/mL)	0.814 ± 0.109	0.815 ± 0.077	0.780 ± 0.086	0.798 ± 0.088
T4(µg%)	3.407 ± 0.560	3.294 ± 0.561	3.395 ± 0.713	3.276 ± 0.496
TSH(pg/mL)	1016.6 ± 365.2	974.1 ± 360.7	934.6 ± 332.0	992.1 ± 341.1

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

Table 13: Number of foetuses (total in group)

Parameter	Group code	Group code	Group code	Group code
	0	125	500	1000
Total number of live foetuses	324	342	305	311
Number of live foetuses – males	167	165	141	143
Number of live foetuses – females	157	177	164	168
Number of dead foetuses	0	0	1	0

 

Table 14: Number of foetuses (average per litter; mean ± SD)

Parameter	Group code	Group code	Group code	Group code
	0	125	500	1000
Total number of live foetuses	14.73 ± 3.27	16.29 ± 2.05	15.25 ± 2.84	15.55 ± 1.43
Number of live foetuses – males	7.59 ± 2.75	7.86 ± 2.22	7.05 ± 2.19	7.15 ± 2.32
Number of live foetuses – females	7.14 ± 2.34	8.43 ± 2.68	8.20 ± 2.44	8.40 ± 1.90
Number of dead foetuses	0.00 ± 0.00	0.00 ± 0.00	0.05 ± 0.22	0.00 ± 0.00

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

 

Table 15: Body weight of foetuses (grams, mean ± SD)

Parameter	Group code	Group code	Group code	Group code
	0	125	500	1000
weight of all foetues	3.82± 0.82	3.45± 0.49	3.71± 0.64	3.55± 0.50
weight of male foetus	3.90± 0.83	3.54± 0.51	3.85 ± 0.64	3.65± 0.52
weight of female foetus	3.73±0.82	3.35± 0.48	3.60 ± 0.64	3.46± 0.50

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

  

Table 16: Mean anogenital distance of foetuses (mm)

Group code	0	0	125	125	500	500	1000	1000
Sex	Male	Female	Male	Female	Male	Female	Male	Female
Mean AGD (mm)	3.45	2.15	3.43	2.19	3.56	2.16	3.47	2.18
Corrected AGD (mm)	2.20	1.40	2.25	1.47	2.28	1.42	2.26	1.45

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

  

Table 17: External alterations - foetuses

Alteration	Group code	Group code	Group code	Group code
	0	125	500	1000
Total number of examined foetuses	324	342	306	311
Total number of examined litters	22	21	20	20
Dead foetus	0	0	1	0
Foetus without tail, absent end part of the spine	0	0	1	0
Number of examined foetuses per litter (mean ± SD)	14.73 ±3.27	16.29 ±2.05	15.30 ±2.81	15.55 ±1.43
Total number of foetuses with alteration	0	0	2	0
Number of foetuses with alteration per litter(mean ± SD)	0.00 ±0.00	0.00 ±0.00	0.10 ±0.31	0.00 ±0.00
Proportion of foetuses with alteration per litter (% mean ± SD)	0.00 ±0.00	0.00 ±0.00	0.67 ±2.07	0.00 ±0.00

 

Table 18: Macroscopic changes of soft tissues – individual foetuses

Alteration	Group code	Group code	Group code	Group code
	0	125	500	1000
Total number of examined foetuses	153	160	141	145
Total number of examined litters	22	21	20	20
With pathological findings - total(number of affected foetuses)	0	0	0	0
Number of examined foetuses in litter (mean ± SD)	6.95 ±1.79	7.62 ±1.02	7.05 ±1.32	7.25 ±0.85
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 19: Skeletal alterations (number of affected foetuses)

Group code		0	125	500	1000
Number of examined foetuses		150	162	156	150
CRANIUM
Nasal bone – i.o.	V	2	0	0	0
Premaxilla – i.o.	V	1	9	0	0
Maxilla – i.o.	V	0	7	0	1
Frontal bone – i.o.	T	6	15	4	5
Parietal bone – i.o.	V	21	74	45	37
Interparietal bone–i.o.	V	64	104	87	60
Supraoccipital bone – i.o.	V	47	101	68	69
Supraoccipital bone – hole	T	72	80	90	79
Supraoccipital bone – b.o.	T	0	3	0	2
Arcus zygomaticus–i.o.	V	29	62	38	40
Squamosal part of temporal bone – i.o.	V	57	103	91	69
Basisphenoid–i.o.	V	2	1	5	2
STERNEBRA
Sternebra – ossification sites – i.o.	V	127	157	142	147
Sternebra – ossification sites – u.	T	40	73	67	77
Sternebra – ossification sites – b.o.	T	1	1	1	2
VERTEBRAE
Vertebrae thoracic centrum – b.o.	T	4	8	10	7
Vertebrae thoracic centrum – b.o., as.os.	T	2	0	1	2
Vertebrae thoracic centrum – d.o.	T	28	51	40	44
Vertebrae thoracic centrum – d.o., as.os.	T	2	2	5	2
Vertebrae lumbar centrum – b.o.	T	0	2	0	0
Vertebrae lumbar centrum – d.o.	T	0	4	3	3
Vertebrae lumbar centrum – d.o., as.os.	T	0	0	1	0
Vertebrae lumbar centrum – misshapen	M	0	0	1	0
Vertebrae lumbar centrum – absent	M	0	0	1	0
Vertebrae sacral centrum – absent	M	0	0	1	0
RIBS
Ribs – supernumerary site	T	22	40	31	30
Ribs – supernumerary full rib	M	0	0	1	0
Ribs – wavy	V	1	22	10	7
Ribs – branched	T	0	0	1	0
SCAPULA
Scapula – i.o	V	22	16	11	16
Scapula – misshapen	T	0	0	1	0

Note:  The results statistically significantly changedon probability level 0.05 were shaded in the summary table No.20.

Table 20: Skeletal alterations(number of affected litters)

 

Group code		0	125	500	1000
Number of examined litters		19	19	19	19
CRANIUM
Nasal bone – i.o.	V	2	0	0	0
Premaxilla – i.o.	V	1	6	0	0
Maxilla – i.o.	V	0	5	0	1
Frontal bone – i.o.	T	3	6	3	3
Parietal bone – i.o.	V	12	15	17	13
Interparietal bone–i.o.	V	16	19	18	18
Supraoccipital bone – i.o.	V	16	19	17	18
Supraoccipital bone – hole	T	16	19	19	19
Supraoccipital bone – b.o.	T	0	1	0	1
Arcus zygomaticus–i.o.	V	13	18	14	13
Squamosal part of temporal bone – i.o.	V	16	19	18	18
Basisphenoid–i.o.	V	2	1	4	2
STERNEBRA
Sternebra – ossification sites – i.o.	V	19	19	19	19
Sternebra – ossification sites – u.	T	14	18	16	18
Sternebra – ossification sites – b.o.	T	1	1	1	2
VERTEBRAE
Vertebrae thoracic centrum – b.o.	T	4	6	7	4
Vertebrae thoracic centrum – b.o., as.os.	T	2	0	1	2
Vertebrae thoracic centrum – d.o.	T	13	16	14	17
Vertebrae thoracic centrum – d.o., as.os.	T	2	2	4	2
Vertebrae lumbar centrum – b.o.	T	0	1	0	0
Vertebrae lumbar centrum – d.o.	T	0	3	3	3
Vertebrae lumbar centrum – d.o., as.os.	T	0	0	1	0
Vertebrae lumbar centrum – misshapen	M	0	0	1	0
Vertebrae lumbar centrum – absent	M	0	0	1	0
Vertebrae sacral centrum – absent	M	0	0	1	0
RIBS
Ribs – supernumerary site	T	11	12	15	12
Ribs – supernumerary full rib	M	0	0	1	0
Ribs – wavy	V	1	9	4	5
Ribs – branched	T	0	0	1	0
SCAPULA
Scapula – i.o	V	9	12	8	9
Scapula – misshapen	T	0	0	1	0

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

Table 21: Skeletal alterations(% proportion of litters with affected foetuses)

Group code		0	125	500	1000
Number of examined litters		19	19	19	19
CRANIUM
Nasal bone – i.o.	V	10.53	0.00	0.00	0.00
Premaxilla – i.o.	V	5.26	31.58	0.00	0.00
Maxilla – i.o.	V	0.00	26.32	0.00	5.26
Frontal bone – i.o.	T	15.79	31.58	15.79	15.79
Parietal bone – i.o.	V	63.16	78.95	89.47	68.42
Interparietal bone–i.o.	V	84.21	100.00	94.74	94.74
Supraoccipital bone – i.o.	V	84.21	100.00	89.47	94.74
Supraoccipital bone – hole	T	84.21	100.00	100.00	100.00
Supraoccipital bone – b.o.	T	0.00	5.26	0.00	5.26
Arcus zygomaticus–i.o.	V	68.42	94.74	73.68	68.42
Squamosal part of temporal bone – i.o.	V	84.21	100.00	94.74	94.74
Basisphenoid–i.o.	V	10.53	5.26	21.05	10.53
STERNEBRA
Sternebra – ossification sites – i.o.	V	100.00	100.00	100.00	100.00
Sternebra – ossification sites – u.	T	73.68	94.74	84.21	94.74
Sternebra – ossification sites – b.o.	T	5.26	5.26	5.26	10.53
VERTEBRAE
Vertebrae thoracic centrum – b.o.	T	21.05	31.58	36.84	21.05
Vertebrae thoracic centrum – b.o., as.os.	T	10.53	0.00	5.26	10.53
Vertebrae thoracic centrum – d.o.	T	68.42	84.21	73.68	89.47
Vertebrae thoracic centrum – d.o., as.os.	T	10.53	10.53	21.05	10.53
Vertebrae lumbar centrum – b.o.	T	0.00	5.26	0.00	0.00
Vertebrae lumbar centrum – d.o.	T	0.00	15.79	15.79	15.79
Vertebrae lumbar centrum – d.o., as.os.	T	0.00	0.00	5.26	0.00
Vertebrae lumbar centrum – misshapen^M	M	0.00	0.00	5.26	0.00
Vertebrae lumbar centrum – absent^M	M	0.00	0.00	5.26	0.00
Vertebrae sacral centrum – absent^M	M	0.00	0.00	5.26	0.00
RIBS
Ribs – supernumerary site	T	57.89	63.16	78.95	63.16
Ribs – supernumerary full rib^M	M	0.00	0.00	5.26	0.00
Ribs – wavy	V	5.26	47.37	21.05	26.32
Ribs – branched	T	0.00	0.00	5.26	0.00
SCAPULA
Scapula – i.o	V	47.37	63.16	42.11	47.37
Scapula – misshapen	T	0.00	0.00	5.26	0.00

Table 22: Skeletal alterations (% proportion of affected foetuses in litter (mean ± SD))

Group code		0	125	500	1000
Number of examined foetuses		150	162	156	150
Number of examined litters		19	19	19	19
CRANIUM
Nasal bone – i.o.	V	1.54 ± 4.65	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Premaxilla – i.o.	V	0.66  ± 2.87	5.70 ± 9.32	0.00 ± 0.00	0.00 ± 0.00
Maxilla – i.o.	V	0.00 ± 0.00	4.46 ± 8.20	0.00 ± 0.00	0.66 ± 2.87
Frontal bone – i.o.	T	4.23  ± 10.77	9.80 ± 15.92	2.56 ± 6.58	3.16 ± 8.03
Parietal bone – i.o.	V	13.71  ± 15.67	46.18 ± 32.78	28.30 ± 18.38	25.51 ± 29.69
Interparietal bone–i.o.	V	42.02 ± 30.21	64.89 ± 28.68	55.03 ± 28.00	41.37 ± 26.57
Supraoccipital bone – i.o.	V	30.39 ± 26.16	63.44 ± 29.67	42.01 ± 25.49	46.97 ± 27.47
Supraoccipital bone – hole	T	46.40 ± 31.62	49.82 ± 21.42	56.90 ± 23.30	53.27 ± 26.81
Supraoccipital bone – b.o.	T	0.00 ± 0.00	1.58 ± 6.88	0.00 ± 0.00	1.32 ± 5.74
Arcus zygomaticus–i.o.	V	21.89 ± 27.99	38.07 ± 21.40	24.36 ± 20.77	25.07 ± 26.14
Squamosal part of temporal bone – i.o.	V	38.43  ± 28.10	63.35 ± 25.53	57.15 ± 29.89	46.84 ± 27.99
Basisphenoid–i.o.	V	1.24 ± 3.73	0.88 ± 3.82	3.25 ± 6.71	1.46 ± 4.48
STERNEBRA
Sternebra – ossification sites – i.o.	V	85.18  ± 22.89	96.78 ± 11.61	89.35 ± 24.11	98.03 ± 4.68
Sternebra – ossification sites – u.	T	26.71  ± 26.31	46.07 ± 28.36	42.71 ± 30.47	53.02 ± 31.49
Sternebra – ossification sites – b.o.	T	0.58 ± 2.55	0.48 ± 2.09	0.58 ± 2.55	1.24 ± 3.73
VERTEBRAE
Vertebrae thoracic centrum – b.o.	T	2.49 ± 4.96	4.73 ± 9.23	6.78 ± 11.31	4.53 ± 10.33
Vertebrae thoracic centrum – b.o., as.os.	T	1.24 ± 3.73	0.00 ± 0.00	0.75 ± 3.28	1.32 ± 3.94
Vertebrae thoracic centrum – d.o.	T	19.08  ± 17.59	30.89 ± 21.10	25.85 ± 20.79	29.81 ± 21.07
Vertebrae thoracic centrum – d.o., as.os.	T	1.24 ± 3.73	1.06 ± 3.20	2.77 ± 5.85	1.32 ± 3.94
Vertebrae lumbar centrum – b.o.	T	0.00 ± 0.00	1.32 ± 5.74	0.00 ± 0.00	0.00 ± 0.00
Vertebrae lumbar centrum – d.o.	T	0.00 ± 0.00	2.49 ± 6.47	1.94 ± 4.65	1.97 ± 4.68
Vertebrae lumbar centrum – d.o., as.os.	T	0.00 ± 0.00	0.00 ± 0.00	0.53 ± 2.29	0.00 ± 0.00
Vertebrae lumbar centrum – misshapen	M	0.00 ± 0.00	0.00 ± 0.00	0.58 ± 2.55	0.00 ± 0.00
Vertebrae lumbar centrum – absent	M	0.00 ± 0.00	0.00 ± 0.00	0.58 ± 2.55	0.00 ± 0.00
Vertebrae sacral centrum – absent	M	0.00 ± 0.00	0.00 ± 0.00	0.58 ± 2.55	0.00 ± 0.00
RIBS
Ribs – supernumerary site	T	14.42 ± 18.95	24.26 ± 31.81	20.62 ± 20.32	19.56 ± 23.69
Ribs – supernumerary full rib	M	0.00 ± 0.00	0.00 ± 0.00	0.58 ± 2.55	0.00 ± 0.00
Ribs – wavy	V	0.58 ± 2.55	14.72 ± 23.40	6.15 ± 12.32	5.65 ± 10.50
Ribs – branched	T	0.00 ± 0.00	0.00 ± 0.00	0.58 ± 2.55	0.00 ± 0.00
SCAPULA
Scapula – i.o	V	14.97 ± 19.26	10.27 ± 10.30	7.05 ± 9.67	10.52 ± 12.46
Scapula – misshapen	T	0.00 ± 0.00	0.00 ± 0.00	0.58 ± 2.55	0.00 ± 0.00

Applicant's summary and conclusion

Conclusions:

In the prenatal developmental toxicity study with HMDTPMP (4-7K) (aqueous solution containing 22.6% w/w active acid), conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal and developmental toxicity was concluded to be greater than 1000 mg active acid/kg bw/day based on no treatment-related adverse effects observed at the highest dose tested.