Trisodium trimetaphosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

One study is available. This study was performed prior to the introduction of GLP.

Link to relevant study records

Reference

Reference 1

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A reproduction study spanning 3 generations of rats reared and maintained on diets containing 0.0% and 0.5% HMP (Hexametaphosphate). Per generation two different litters were produced; one was sacrificed and the other was bred to produce the next generation. Matings were carried out between 16 females and 8 males in each group when the rats were 100 days old. Observations were limited to: the numbers of pups born, pup mortality up to 21 days, organ weights and histological changes of tissues and organs.

GLP compliance:

no

Remarks:

study predates GLP

Limit test:

no

Species:

rat

Strain:

other: Rochester (Ex-Wistar 1923)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: The original groups of rats were mated at 100 days
- Weight at study initiation: Bodyweight was approximately 70 grams for males and females at the start of the 100 day exposure.
- Fasting period before study:
- Housing: cages
- Diet (e.g. ad libitum): ad libitum,
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
no data

IN-LIFE DATES: From: *** To: ***

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION

Diet mixtures were prepared using a basal ration of Purina Fox Chow Meal into which the appropriate amounts of sodium hexametaphosphate were mixed by a mechanical mixer. At weekly intervals. Diets were stored during the week in galvanized iron pails with covers.

Details on mating procedure:

- M/F ratio per cage: 1male: 2 female
- Length of cohabitation: 7 days
- Proof of pregnancy: no data
- Unsuccessful pairing replacement of first male by another male with proven fertility?: no.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): no data
- other: males were rotated so that at each mating a different male would be placed in the cage with the female.

Details on mating schedules are tabulated below. - Table 1.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

Exposure took place for 100 days prior to mating, 21 days of gestation and during lactation for the P1, F1b, F2b. The F3b were exposed for their first 21days after which time they were sacrificed. Litters were culled to ten pups with approximately 5/sex/group on postnatal day 5.
For the F1, F2, F3-generations, offspring were weaned and exposed during an interim period of 10 days and then during a mating period of 7 days. Dams were exposed continually during gestation and lactation. Offspring of the second litter were again mated for 7 days after a 10 day interim period following weaning. The first litters (F1a, F2a, F3a) were sacrificed at 30 days of age. The F1c and F3b litters were sacrificed after 21 days.

Frequency of treatment:

daily in feed

Details on study schedule:

- F1 parental animals were mated at 100 days of age in order to produce the sacrificial group F2a, and at 151 days F1 animals were mated to produce the next parental generation for F2b.
- Per female the litters were culled to ten pups with approximately 5/sex/group. There are no details on the selection of parental animals.
- Age at mating of the mated animals in the study: 100 days to produce the sacrifical litter and at 151 days to produce the next parental generation.

More details on protocol are tabulated below. - Table 1.

Remarks:

Doses / Concentrations:
0.0% and 0.5%
Basis:
nominal in diet

No. of animals per sex per dose:

16 females and 8 males were used to produce the subsequent generation. P1 , F1b , F2b , F3b.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Two doses were considered; 0.05% and 0.5%. After 100 days on these diets the 0.5% group exhibited no depression of body weight and was selected for use in this study.
- Other: The first litters born per generation were sacrificed, the F1a, F2a, F3a. The second litters born in a generation, the F1b, F2b, were mated to produce the next generation. The F1c was an additional litter produced and treated the same as the F1a.

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured weekly during the 100 day pre-mating exposure for each generation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not measured

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not measured

Oestrous cyclicity (parental animals):

not evaluated

Sperm parameters (parental animals):

not evaluated

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 5 postpartum: yes
- excess pups were sacrificed to leave 10 per litter.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number of pups, pups/litter, pup mortality up to 21 days of age, weight gain per litter]

GROSS EXAMINATION OF DEAD PUPS: No

Postmortem examinations (parental animals):

SACRIFICE
- The first litters prodced per generation, the F1a, the F2a and the F3a were sacrificed at 30 days of birth.
- There is no data on the fate of the parents after they were mated to produce each generation.

GROSS NECROPSY
no data

HISTOPATHOLOGY
No histopathological examinations were performed for parental rats.

ORGAN WEIGHTS
No organ weight analyses were performed for parental rats.

Postmortem examinations (offspring):

SACRIFICE
- The non-parental litters, F1a , F1c, F2a , F3a , and F3b were sacrificed after 21 days.
- Observations amongst these litters were limited to litter weights on the 21st day. In the F3b histopathological and organ weight examinations were performed.

GROSS NECROPSY
F3b examined only.

HISTOPATHOLOGY / ORGAN WEIGTHS
The ten males and ten females from final litter, the F3b litter, were evaluated for body weight, histopathology (liver, kidneys, gonads, lungs, brain, stomach, heart, spleen, adrenal, stomach, large and small intestines, bladder, lymph nodes, gut, marrow, pancreas) and organ weights (liver, kidneys, testes, lungs, brain, stomach, heart, spleen).

Statistics:

no data

Reproductive indices:

no data

Offspring viability indices:

no data

Clinical signs:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

In Table 2. a summary is presented of the reproductive performance.
Number of females mated: In all cases 15 or 16 females were mated after a 7-day cohabitation with one male (1 male caged with 2 females).
Number of pregnancies: Most of the pregnancies were successful. For the control rats there were 10 to 15 pregnancies per litter and for the rats maintained on the diet containing 0.5% HMP there were 10 to 13 in each mating.
Number of rats born: Comparable between the control and 0.5% group, the average number of pups ranged from 8-10 in both the controls and 0.5% group.

Key result

Dose descriptor:

NOAEL

Effect level:

0.5 other: %

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: dose level calculated

Key result

Critical effects observed:

no

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
The number of pups surviving at 21 days was 65-122 in the control groups and 69-111 in the 0.5% HMP group.
The average weight of the pups at 21 days was 41.8 - 48.7 g in the control groups and 40.9 - 45.4g in the 0.5% HMP group.

BODYWEIGHT (OFFSPRING)
After 21 days the newly born rats surviving to 21 days were weighed per litter, differences between the control and HMP-fed rats were not considered to be significant. See Table 2.

F1a - The average bodyweight of rats on the HMP diet was less than that of the control group.
F1b - The average bodyweight of rats on the HMP diet was less than that of the control group.
F1c - The average bodyweight of rats on the HMP diet was greater than that of the control group - based on this the authors considered the decreased average litter weights of the F1a and F1b to be mere coincidental variations.

F2a - The average bodyweight of rats on the HMP diet was less than that of the control group.
F2b - There was a small difference, less than a gram, in the average weight of the control and HMP-fed rats. There was a peculiar delay of the HMP-fed rats within the first week on the diet, but growth was rapidly recovered and surpassed that of the control group.

F3a - The average weight of the HMP-fed and control rats was almost identical at 42.5 and 42.4 gram respectively
F3b - The average weight of the MHP-fed rats was greater than that of the controls.

ORGAN WEIGHTS (OFFSPRING)
When the F3b rats were at weaning age they were sacrificed. The author described the organ weights and organ weight body weight ratios as, being within normal ranges. See Table 3

HISTOPATHOLOGY (OFFSPRING)
No histological changes were found that were attributed to the presence of HMP in the diet. No tumours were present and only normal tissues were found.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.5 other: % w/w

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: dose level calculated

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

0.5 other: % w/w in diet

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: dose level calculated

Key result

Key result

Reproductive effects observed:

no

Table 2. Reproductive performance

Generation & Litter	F1a	F1b	F1c	F2a	F2b	F3a	F3b
Controls
Females	16	15	15	16	15	16	16
Pregnancies	14	10	10	11	10	15	11
Total pups	107	95	77	94	93	145	115
Avg. pups/litter	7.6	9.5	9.6	8.5	9.3	9.7	10.4
Mortality (days 0-5)	0	1	3	0	10	8	10
Mortality (days 6-21)	0	1	5	3	4	9	6
Total rats surviving to day 21	107	84	65	87	77	122	90
Avg. weight of surviving rats	48.7	45.2	42.0	44.9	41.8	42.4	42.9
0.5% Hexametaphosphate group
Females	16	15	15	16	16	16	16
Pregnancies	11	10	13	13	10	12	10
Total pups	83	100	126	118	89	119	97
Avg. pups/litter	8.3	10.0	9.7	9.1	8.9	9.9	9.7
Mortality (days 0-5)	6	15	3	11	2	1	3
Mortality (days 6-21)	5	1	0	9	11	3	0
Total rats surviving to day 21	69	77	111	95	73	108	87
Avg. weight of surviving rats	43.1	43.5	44.7	44.3	40.9	42.5	45.4

Table 3 - Organ Weights

Group	No. of Rats	Body Wt.	Liver	Kidneys	Testes	Lungs	Brain	Stomach	Heart	Spleen
Control Males	10	111	4.84	1.28	1.20	0.80	1.59	1.04	0.55	0.59
0.5%HMP-fed males	10	102	4.74	1.16	1.04	0.82	1.49	1.07	0.56	0.37
Control Females	10	96	4.03	1.06	n/a	0.69	1.43	1.02	0.53	0.64
0.5%HMP-fed females	10	90	3.71	1.03	n/a	0.67	1.54	1.09	0.47	0.36

Conclusions:

Three generations of rats reared and maintained on diets containing 0.5% HMP showed no adverse effects to the test material - Control and test data were comparable across the range of observations made. Observations were limited to reproductive performance, pup mortality, organ weights and tissue and organ histopathology. Based on this evidence sodium metaphosphate is not considered to be classified as a reproductive toxicant according to Regulation (EC) 1272/2008 (EU CLP).

Although this study is limited and deficient by modern standards, it is still possible to make some valid scientific conclusions from the data. Adequate data are reported to show no effects on fertility or reproductive performance or on offspring growth and development over three generations with two litters per generation. Thus, the study should be considered acceptable for the dose level tested, 0.5% test material in the diet.

Reference 2

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Well documented study with the following deviations: Only one dose level was included in the study. Test material was not analyzed in diet. No impurity profile of test material was provided. Source of animals was not specified. The group size was smaller than the 20 females and 10 males required in OECD guideline 416. There was no recorded acclimation period for the P generation. Only one dose tested. Daily observation of clinical signs of parents and observations of abnormal behaviour of offspring are not reported. Body Weights: parental body weights were not recorded during gestation and lactation. Food consumption was not determined. Estrus cycle was not evaluated. Sperm parameters were not included. Offspring sex and sex ratios were not determined. Litter body weight was reported; however, individual pup body weights were not recorded. There is no report of gross pathology and necropsy observations. Organ weight data for the uterus, ovaries, epididymides, prostate, seminal vesicles, pituitary gland, adrenals and thyroid gland were not included. Histopathology was conducted only on the F3b offspring and did not include the following organs: uterus, cervix, vagina, epididymides, seminal vesicles, prostate, coagulating gland, pituitary gland. Note that the known target organ (kidney) was evaluated. Statistics are not reported. This study is considered to fulfil the data requirements as a supporting study for REACH.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Three generations of rats were reared on diets containing 0 and 0.5% sodium tripolyphosphate. Two litters were examined in each generation. Matingwas carried out between 16 females and 8 males of each group when the rats were 100 days old.

GLP compliance:

no

Remarks:

Study predates GLP

Limit test:

no

Species:

rat

Strain:

other: Rochester Strain (Ex-Wistar 1923)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 60 g
- Diet: e.g. ad libitum
- Water :e.g. ad libitum

Route of administration:

oral: feed

Details on exposure:

Exposure took place for 100 days prior to mating, 21 days of gestation and during lactation. For the F1, F2, F3-generations, offspring were weaned and exposed during an interim period of 10 days and then during a mating period of 7 days. Dams were exposed continually during gestation and lactation. Offspring of the second litter were again mated for 7 days after a 10 day interim period following weaning. The first litters (F1a, F2a, F3a) were sacrificed at 30 days of age. The second litters (F1b, F2b, F3b) were used to produce the succeeding generation.

Details on mating procedure:

Each male was caged with two females for 7 days until conception occurred.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure took place for 100 days prior to mating, 21 days of gestation and during lactation. For the F1, F2, F3-generations, offspring were weaned and exposed during an interim period of 10 days and then during a mating period of 7 days. Dams were exposed continually during gestation and lactation. Offspring of the second litter were again mated for 7 days after a 10 day interim period following weaning. The first litters (F1a, F2a, F3a) were sacrificed at 30 days of age. The second litters (F1b, F2b, F3b) were used to produce the succeeding generation.

Remarks:

Doses / Concentrations:
0.5% in diet
Basis:
nominal in diet

No. of animals per sex per dose:

P1, F1, F2, F3: 16 females and 8 males

Control animals:

yes, concurrent vehicle

Sperm parameters (parental animals):

Not evaluated

Litter observations:

number of pups, stillbirths, live births, weight gain per litter
Not Evaluated: sex of pups, presence of gross anomalies
physical or behavioural abnormalities, , no individual pup body weights

Postmortem examinations (offspring):

F3b generation groups of male (8/group) and female (16/group) rats were fed control diet or diet with 0.5% test material for 100 days (P1) generation. Body weights were measured weekly during the 100 day pre-mating exposure for each generation. Each male was caged with two females for 7 days until conception occurred. Pregnancy rates were determined. The first litter born of the parental animals was designated as the F1a litter. Upon delivery, offspring were counted and average litter weights determined. On day 5 pups were culled to 10 per litter. Body weights per litter were determined on day 21. Pup survival was determined on days 1-5 and days 6-21. The F1a litter was sacrificed at 30 days postnatally. Parental animals were mated again 10 days after the sacrifice of the F1a litter. Mating took place for 7 days. A second litter (F1b) was born after approximately 21 days of gestation. Upon delivery, offspring were counted and the average litter weights determined. On day 5 pups were culled to 10 per litter. Body weights per litter were determined on day 21. Pup survival was determined on days 1-5 and days 6-21. Following weaning of the F1b litter, 8 males and 16 females for the control group and the treated group were fed their respective diets for 100 days. Again two litters (F2a and F2b) were produced. Parental animals were mated for 7 days following 10 days after the sacrifice of the F2a litter (pups were 30 days old). Following birth of the F2b litter the same offspring and parental parameters were measured. Eight males and 16 females of the F2b litter were designated for continual exposure for another 100 days. Upon completion of the exposure of the F2b litter, animals were mated again to produce the F3a and F3b litters in the same manner described above. The ten males and ten females from final litter, the F3b litter, were evaluated for body weight, histopathology (liver, kidneys, gonads, lungs, brain, stomach, heart, spleen, adrenal, stomach, large and small intestines, bladder, nodes, marrow, pancreas) and organ weights (liver, kidneys, testes, lungs, brain, stomach, heart, spleen). Individual animal data were collected and recorded throughout the study.
Organ weights:
liver, kidneys, gonads, lungs, brain, stomach, heart, spleen on F2b offspring.
Not evaluated: Uterus, ovaries, epididymides (total and cauda), prostate, seminal vesicles, pituitary, thyroid, adrenal glands
Histopathology:
Kidneys, heart, brain, spleen, lungs, ovaries, testes, adrenal, bladder, lymph nodes, gut, bone marrow, pancreas on F3b animals.
Not Evaluated: Vagina, uterus with cervix, epididymis, seminal vesicle, prostate coagulating gland

Key result

Dose descriptor:

NOAEL

Effect level:

0.5 other: % w/w

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: dose level calculated

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.5 other: % w/w

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: calculated

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

0.5 other: % w/w in diet

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: calculated

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

This study included only one dose level of the test material, 0.5% in the diet. 

NOAEL calculated in accorance with Appendix F, guidelines for the preparation of toxicological working papers for the joint FAO/WHO expert committee on food additives, December 2000.

There were occasional fluctuations in the fertility index.  The lower fertility index in the F2a, F3a and F3b litters is considered spurious and not treatment-related.  The fertility indices of the treated group were greater than the control group for some litters (F1a, F2b); the same as the control group for the F1b litter, and less than the control group for other litters (F2a, F3a, F3b).  These variations are not considered to show a treatment-related effect on fertility.  The duration of pregnancy, live birth index, litter size, litter weights, survival index, viability index and lactation index were all comparable between control and treated groups.  Organ weights were similar between control and treated groups.  There were no treatment-related histopathological findings.

Parameter			Control		High dose
		Generation	M	F	M	F
Mortality	Incidence	P	0	0	0	0
		F1	0	0	0	0
		F2	0	0	0	0
		F3	0	0	0	0
Food consumption	% of control		NA	NA	NA	NA
Body weight gain	% of control		100	100	100	100
Clinical Observations specify effects	Incidence		NA	NA	NA	NA
Organ weights (F3b)	% of control		95 -103	95 -103	95 -103	95 -103
Pathology
Histopathologic examination: F3b Immature testes Renal cyst Parasites in intestine Focal pulmonary hemorrhage Parasites in renal pelvis	Incidence		10 - - - -	- - 1 1 -	10 1 1 1 -	- - - - 2
Reproductive Performance
Mating index: P			-	NA		NA
F1			-	NA	-	NA
F2			-	NA	-	NA
F3			-	NA	-	NA
Fertility index: P, la			-	87.5	-	93.8
F1b			-	75	-	75
F2a			-	82.3	-	68.8
F2b			-	76.5	-	81.3
F3a				81.3	-	68.8
F3b				80	-	62.5
Number of implantation sites	Mean		-	NA	-	NA
Duration of pregnancy: P, 1a	Mean days			24.7		24.06
F1b				25.1	-	25.25
F2a				23.36	-	24.91
F2b				25	-	24.4
F3a				25.8	-	25.1
F3b				25.2	-	25
Birth index
Live birth index: P, 1a			-	93.8	-	100
F1b				93.8	-	100
F2a				100	-	100
F2b				100	-	93.8
F3a				93.8	-	100
F3b				100		87.5
Gestation index
Litter size: P, 1a	Mean		-	8.9	-	9.2
F1b				11	-	13
F2a				9.2	-	9.5
F2b				10.7	-	10.2
F3a				10.4	-	10.2
F3b				8.4	-	10.5
Litter weight: P, 1a	Mean, day 21, g		-	307	-	283
F1b				348	-	392
F2a				279	-	341
F2b				379	-	325
F3a				270		349
F3b				319	-	388
Pup weight, 1a	Mean: day 21, g			36.8	-	37.8
F1b				42.2	-	40.5
F2a				42.9	-	42.1
F2b				40.3	-	41.5
F3a				41.4	-	43.7
F3b				45.6	-	41.9
Sex ratio	Male/female			NA		NA
Survival index: P, 1a	Day 5			91.4	-	95.6
F1b				91.6	-	97.4
F2a				93.3	-	88.6
F2b				96.7	-	93.4
F3a				82.4	-	85.2
F3b				74.8	-	98.8
Viability index: P, 1a	Day 21		-	86.2	-	81.1
F1b				75	-	74.3
F2a				74.2	-	84.8
F2b				85	-	77
F3a				72.8	-	72
F3b				76.2	-	88
Lactation index: P, 1a	Day 21			86.2	-	81.1
F1b				75	-	74.3
F2a				74.2	-	84.8
F2b				85	-	77
F3a				72.8	-	72
F3b				76.2	-	88
Sperm characterization				NA		NA
Number	% of control			NA		NA
Deformations	% of control			NA		NA

Conclusions:

The study is relevant to the hazard evaluation of STPP, but limited in data on the endpoints tested because only one dose was included. In addition, histopathological evaluation of all major reproductive organs, except testes and ovaries, was not conducted. Therefore, this study is considered to be Klimisch code 2: reliable with restrictions. Although the restrictions are significant, the investigation is well documented and scientifically acceptable. Reliable conclusions can be drawn from this study based on the data reported with consideration of the limitations. It can be concluded that STPP did not induce adverse effects on fertility and reproduction in rats when fed for three generations at a level of 0.5% in the diet. This no-effect level provides a basis for some risk assessment even though not all parameters currently included in the OECD 416 guideline were evaluated. Key Restrictions: Only one dose level was tested and important data on the reproductive organs were not collected; no evaluation of some sex organs, no sperm evaluations, individual pup data not evaluated, Specific deficiencies may be addressed by reviewing other studies on STPP and on similar inorganic phosphates. Together, the data on several phosphates indicate no major concerns regarding reproductive or fertility.

Reference 3

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Well documented study with the following deviations: Only one dose level was included in the study. Test material was not analyzed in diet. No impurity profile of test material was provided. Source of animals was not specified. The group size was smaller than the 20 females and 10 males required in OECD guideline 416. There was no recorded acclimation period for the P generation. Only one dose tested. Daily observation of clinical signs of parents and observations of abnormal behaviour of offspring are not reported. Body Weights: parental body weights were not recorded during gestation and lactation. Food consumption was not determined. Estrus cycle was not evaluated. Sperm parameters were not included. Offspring sex and sex ratios were not determined. Litter body weight was reported; however, individual pup body weights were not recorded. There is no report of gross pathology and necropsy observations. Organ weight data for the uterus, ovaries, epididymides, prostate, seminal vesicles, pituitary gland, adrenals and thyroid gland were not included. Histopathology was conducted only on the F3b offspring and did not include the following organs: uterus, cervix, vagina, epididymides, seminal vesicles, prostate, coagulating gland, pituitary gland. Note that the known target organ (kidney) was evaluated. Statistics are not reported. Study has been reviewed by an expert assessor (see expert report attached) and determined to be suitable for use for the purposes of REACH registration.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of male (8/group) and female (16/group) rats were fed control diet or diet treated with 0.05% test material for 100 days (P1) generation. Body weights were measured weekly during the 100 day pre-mating exposure for each generation. Each male was caged with two females for 7 days until conception occurred. Pregnancy rates were determined. The first litter born of the parental animals was designated as the F1a litter. Upon delivery, offspring were counted and average litter weights determined. Pup survival was determined on days 1-5 and days 6-21. On day 5, pups were culled to 10 per litter. Body weights per litter were determined on day 21. The F1a litter was sacrificed at 30 days postnatally. Parental animals were mated again 10 days after the sacrifice of the F1a litter (ten-day interim period). Mating took place for 7 days. A second litter (F1b) was born after approximately 21 days of gestation. Upon delivery, offspring were counted and the average litter weights determined. Pup survival was determined on days 1-5 and days 6-21. On day 5 pups were culled to 10 per litter. Body weights per litter were determined on day 21. Following weaning of the F1b litter, 8 males and 16 females from the F1b litter from the control group and the treated group were fed their respective diets for 100 days. Again two litters (F2a and F2b) were produced in the same sequence described for the F1 litters. Parental animals were mated for 7 days after the ten-day interim period after the sacrifice of the F2a litter (pups were 30 days old). Following birth of the F2b litter the same offspring and parental parameters were measured. Eight males and 16 females of the F2b litter were designated for continual exposure for another 100 days. Upon completion of the exposure of the F2b litter, animals were mated again to produce the F3a and F3b litters in the same manner described above. The ten males and ten females from the final litter, the F3b litter, were evaluated for body weight, histopathology (liver, kidneys, gonads, lungs, brain, stomach, heart, spleen, adrenal, stomach, large and small intestines, bladder, bone marrow, muscle, pancreas, lymph nodes, gut) and organ weights (liver, kidneys, testes, lungs, brain, stomach, heart, spleen). Individual animal data were collected and recorded throughout the study.

GLP compliance:

no

Remarks:

study predates GLP

Limit test:

yes

Species:

rat

Strain:

other: Rochester Strain (Ex-Wistar 1923)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: Body weight was approximately 60 grams for males and 59 grams for females at the start of the 100 day exposure.
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not applicable

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on exposure:

No data

Details on mating procedure:

- Length of cohabitation: 7 days
- Any other deviations from standard protocol: Litters were culled to ten pups on day 5 of lactation.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

Exposure took place for 100 days prior to mating, 21 days of gestation and during lactation. Litters were culled to ten pups with approximately 5/sex/group on postnatal day 5. For the F1, F2, F3-generations, offspring were weaned and exposed during an interim period of 10 days and then during a mating period of 7 days. Dams were exposed continually during gestation and lactation. Offspring of the second litter were again mated for 7 days after a 10 day interim period following weaning. The first litters (F1a, F2a, F3a) were sacrificed at 30 days of age. The second litters (F1b, F2b) were used to produce the succeeding generation. The F3b pups were used for the histopathology evaluation and organ weight determinations.

Frequency of treatment:

daily, ad libitum

Details on study schedule:

Beginning with the original groups of rats, 16 female and 8 male rats were removed at the age of 100 days and mated. The matings were carried out in such a way that male rats were rotated through cages each housing two female rats.

For the F1, F2, F3-generations, offspring were weaned and exposed during an interim period of 10 days and then during a mating period of 7 days.
Offspring of the second litter were again mated for 7 days after a 10 day interim period following weaning.

The first litters (F1a, F2a, F3a) were sacrificed at 30 days of age. The second litters (F1b, F2b) were used to produce the succeeding generation. The F3b pups were used for the histopathology evaluation and organ weight determinations.

Litters were culled to ten pups with approximately 5/sex/group on postnatal day 5.

Dose / conc.:

0.05 other: % w/w nominal in diet

No. of animals per sex per dose:

P1, F1, F2, F3: 16 females and 8 males

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The reproduction study began with 16 female and 8 male rats from the control diet group and from the group given the diet containing 1% of TMP in the 2-year study. When it was discovered that the male rats given a 1% Trimetaphosphate diet were not maintaining normal growth, it was obvious that this level was unsuitable one for reproduction study. Consequently additinoal diet groups were started at 0.1% and at 0.05% to be sure that no detectable effect could be laid to the TMP in the diet. The continued stress of repeated production of generations and litters of rats could then be tested. In 1957 it was decided to conduct a reproduction study on rats maintained on the diet containing 0.05% trimetaphosphate.

- Rationale for animal assignment (if not random): no data

- Other: no data

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data
- The animals were monitored for general well-being.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the 100 day pre-mating period, but not evaluated during gestation and lactation.

Oestrous cyclicity (parental animals):

Not evaluated.

Sperm parameters (parental animals):

The following were not evaluated: testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.

Litter observations:

number of pups, stillbirths, live births, weight gain per litter

The following was not evaluated: sex of pups, presence of gross anomalies, physical or behavioural abnormalities. No individual pup body weights.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals.
- Maternal animals: All surviving animals.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

ORGAN WEIGHTS; F3b:
liver, kidneys, testes, lungs, brain, stomach, heart, spleen on 10/sex/group from the F3b offspring at 21 days of age.
The following was not evaluated: Uterus, ovaries, epididymides (total and cauda), prostate, seminal vesicles, pituitary, thyroid, adrenal glands

HISTOPATHOLOGY; F3b:
Kidneys, heart, brain, spleen, lungs, liver, ovaries, testes, adrenal, urinary bladder, stomach, large and small intestines, muscle, bone marrow, muscle, pancreas, gut and lymph noted on F3b animals.
The following was not evaluated: Vagina, uterus, epididymis, seminal vesicle, prostate
coagulating gland.
Not evaluated

Key result

Dose descriptor:

NOAEL

Effect level:

0.05 other: % w/w

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects noted

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects noted

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.05 other: % w/w

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects notes

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects noted

Remarks on result:

other: calculated

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

0.05 other: % w/w

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects noted

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects noted

Remarks on result:

other: calculated

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

NOAEL calculated in accorance with Appendix F, guidelines for the preparation of toxicological working papers for the joint FAO/WHO expert committee on food additives, December 2000.

This study included only one dose level of the test material, 0.05% in the diet. Table 1 summarizes results on many parameters. There was no treatment-related effect on litter weights. The fertility index, duration of pregnancy, live birth index, litter size, litter weights, average pup weights, number of pups per litter, survival index, viability index and lactation index were all comparable between control and treated groups. Organ weights were similar between control and treated groups. Treated females of the F3b generation had somewhat lower spleen weights than control animals. There were no treatment-relatedhistopathologicalfindings.

 

Table 1. Reproductive toxicity

Parameter			Control		High dose
		Generation	M	F	M	F
Mortality	Incidence	P	0	0	0	0
		F1	3	1	0	0
		F2	0	2	0	0
		F3	0	1	0	1
Food consumption	% of control		NA	NA	NA	NA
Body weight gain	% of control		100	100	100	100
Clinical Observations specify effects	Incidence		NA	NA	NA	NA
Organ weights (F3b) Liver Kidneys Testes Lungs Brain Stomach Heart Spleen	% of control				95.6 98.9 92.1 95.4 100.6 88.0 89.4 83.7	96.8 91.9 - 93.4 105.5 92.5 90.5 69.2
Pathology
Histopathologicexamination: F3b	Incidence	0	0	0	0	0

 

Conclusions:

The test material did not cause any adverse effects on fertility, reproductive performance, offspring viability, offspring survival and offspring body weight in male and female rats when administered continuously for three generations in the diet at a dose level of 0.05% in the diet. The test material did not cause any treatment-related effects on organ weights or any histopathological findings on the tissues evaluated at a dose level of 0.05% in the diet.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The dataset has been reviewed by an external assessor (M Weiner, TOXpertise, LLC) and it is concluded that the data is adequate and reliable for use as a key study under REACH. Please see expert report attached in endpoint under 'background information'.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The above NOAEL is based on the highest concentration tested in the study.

The Hodge reproduction study (1960) was conducted prior to the institution of good laboratory practice guidelines and to the current OECD Guideline 416. Therefore, the study has deficiencies when examined according to today’s standards. The study is considered a Klimisch Code 2, reliable with restrictions (see report attached in study summary). The conclusions regarding the hazard identification of trisodium trimetaphosphate are supported by data on sodium tripolyphosphate / pentasodium triphosphate (STPP) and sodium hexametaphosphate (SHMP), both of which are structurally similar to trisodium trimetaphosphate. All three inorganic phosphate salts had no adverse effects on reproduction and fertility when tested in rats over three generations (Hodge 1959, 1960).

 

Based on this evidence trisodium trimetaphosphate is not considered to be classified as a reproductive toxicant according to Regulation (EC) 1272/2008 (EU CLP).

Short description of key information:
Regulation (EC) No. 1907/2006 - Annex VIII - Section 8.7.1. Screening for reproductive toxicity.
A Column 2 adaptation has been used to waive the need to conduct this study - Screening for reproductive toxicity does not need to be conducted if a study is available for a two-generation reproductive toxicity study (Annex IX, Section 8.7.3). A multi-generation reproductive toxicity study can be found in Section 7.8.1 of this dossier.

Regulation (EC) No. 1907/2006 - Annex IX - Section 8.7.3. Two generation reproductive toxicity study.
The rats were fed diets containing 0.05% trisodium trimetaphosphate during the 100 days prior to mating, during the 21 days of gestation and during the lactation for the P1, F1b, F2b. The F3b were exposed for their first 21days after which time they were sacrificed.
Observations were limited to body weight, histopathology (liver, kidneys, gonads, lungs, brain, stomach, heart, spleen, adrenal, stomach, large and small intestines, bladder, lymph nodes, gut, marrow, pancreas) and organ weights (liver, kidneys, testes, lungs, brain, stomach, heart, spleen).
The study was conducted before the introduction of GLP and the modern standard test Guideline OECD 416. This result provides evidence for the assumption that there is no concern with regard to effects of trisodium trimetaphosphate on reproduction.

Justification for selection of Effect on fertility via oral route:
The dataset has been reviewed by an external assessor (M Weiner, TOXpertise, LLC) and it is concluded that the data is adequate and reliable for use as a key study under REACH. Please see expert report attached in endpoint under 'background information'.

Effects on developmental toxicity

Description of key information

Regulation (EC) No. 1907/2006 - Annex VIII - Section 8.7.1. Screening for developmental toxicity.
A Column 2 adaptation has been used to waive the need to conduct this study - Screening for developmental toxicity does not need to be conducted if a study is available for pre-natal developmental toxicity (Annex IX 8.7.2). An Annex IX 8.7.2 study can be found in section 7.8.2 of this dossier.
Regulation (EC) No. 1907/2006 - Annex IX - Section 8.7.2. Prenatal developmental toxicity
No pre-natal developmental toxicity study is submitted on the basis that the substance is considered to be of low toxicological activity. It is therefore not considered to be ethically justified to conduct additional in vivo studies. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Trisodium trimetaphosphate is not considered to be a reproductive or developmental toxicant in accordance with Regulation (EC) No.1272/2008 (EU CLP).