Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

- Experimental in vivo study (two-generation reproductive toxicity study, OECD Guideline 416) on the analogue substance “ bis[tetrakis(hydroxymethyl)phosphonium] sulphate”, CAS Number: 55566-30-8 at 78%. By analogy, the test substance is not considered as reprotoxic and not classified according to EU criteria.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Deviations from the current OECD 416 (22/01/2001) test guideline: Only 100 instead of 200 individual sperm cells per animal were assessed for morphological changes. Brain, spleen and thymus were not weighed in any of the F1 and F2 pups selected for necropsy. These deviations did not adversely affect the study results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Only 100 instead of 200 individual sperm cells per animal were assessed for morphological changes. Brain, spleen and thymus were not weighed in any of the F1 and F2 pups selected for necropsy. These deviations did not adversely affect the study results.
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
other: Japanese ministry of Agriculture, Forestry and Fisheries testing Guidelines for Toxicology studies, 59 NohSan No. 4200
GLP compliance:
yes (incl. certificate)
Remarks:
1998-07-21
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sprague-Dawley Crl:CD(SD)IGS BR, from Charles River Limited, UK
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 188-229 g, Females: 131-176 g
- Housing: four by sex, then one male with one female during mating, then males returned to original cages and mated females are caged individually
- Use of restrainers for preventing ingestion (if dermal): yes/no
- pelleted diet and tap water ad libitum
- Acclimation period:14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23°C
- Humidity: 40-70%
- Air changes: at least 15 per hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: dosing phase from 10 February to 4 November 1998
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared daily in distilled water.
The high dose level was prepared by weighing an aliquot of test material and adding the appropriate volume of vehicle.
The intermediate and low doses were prepared by taking a subsample of the high dose formulation and mixing with appropriate
volumes of vehicle.

VEHICLE
- Concentration in vehicle: 0.1, 0.75 and 1.5 mg/mL
- total volume applied: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 13 days for P generation and 17 days for F1 generation
- Proof of pregnancy: Following pairing, a vaginal smear was prepared for each female. The presence of sperm within the vaginal smear
and/or vaginal plug in situ was taken as positive evidence of mating.
- After successful mating each pregnant female was caged individually
- parturition time: Each Parental and F1 female was observed at 0830, 1230 and 1630 hours at or around the period of expected
parturition. At weekends, observations were carried out at 0830 and 1230 hours only.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Substance content was checked once per week for the first 4 weeks, approximately once per month thereafter and always found
to be in the range of +/- 10% of the nominal value, except on Day 87, when the concentration of the high dose level was 112% of the
nominal value.
Duration of treatment / exposure:
Exposure period: Maximum total exposure : P/males : (76+13): 89 days P/females : (76+13+25+21): 135 days F1/males : (21+82+17): 120 days F1/females : (21+82+17+25+21): 166 days Premating exposure period (males): P males : 70 days. F1 males : 82 days Premating exposure period (females): P females : 70 days. F1 females : 82 days. Duration of test: 9 months (in-life phase of the study).
Frequency of treatment:
Daily, 7 days per week
Details on study schedule:
- F1 parental animals not mated until 17 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: no data
Remarks:
0.78, 5.85 and 11.7 mg/kg bw (expressed as main ingredient)
Basis:
other: 1.0, 7.5, 15.0 mg/kg bw/day, (expressed as active substance, THPS 78%)
No. of animals per sex per dose:
32 males and 32 females in both P and F1 generations group
Control animals:
yes, concurrent vehicle
Details on study design:
Duration of the treatment:
Following a 14 day acclimatisation period, Parental males and females were dosed for 76 days prior to pairing and for up to 13 days
during mating. The test material was then administered only to females during gestation and lactation phases until Day 21 post partum.
Selected F1 animals were dosed from Day 21 post partum for 82 days prior to pairing, then for up to 17 days during mating.
The test material was then administered only to females during gestation and lactation phases until Day 21 post partum.
P / males : 89 days
P / females : 135 days
F1 / males :120 days
F1 / females : 165 days
Positive control:
none
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: twice daily

MORTALITY: twice daily, daily at week-ends and holidays

BODY WEIGHT: see details in table 1 below

FOOD CONSUMPTION: see details in table 2 below
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily for 14 days prior to mating for Parental and F1 females. Smears were stained with Giemsa stain.
Oocytes parameters: for P and F1 females of control and high dose groups:
- Number
- Follicle size (small, medium, large)

Sperm parameters (parental animals):
For P and F1 males of all dose groups:
- Motility
- Concentration
- Morphology (100 individual sperm)
Litter observations:
STANDARDISATION OF LITTERS
On Day 4 post partum, litter sizes of both generations were standardised to 8 pups per litter, 4 males and 4 females if possible. Offspring were selected randomly, using random number tables. Litter sizes of 8 or less on day 4 post partum were not standardised.
Postmortem examinations (parental animals):
SACRIFICE: All surviving animals, following successful weaning of the offsprings from the P and F1 matings

GROSS NECROPSY: for all animals of the Parental and F1 generations

HISTOPATHOLOGY
All adult animals from control and high dose groups of the Parental and F1 generations:
organs: ovaries, uterus, cervix, vagina, testes, epididymides, seminal vesicles, prostate, coagulating gland, pituitary gland, significant
abnormalities and liver (potential target organ examined also in low and mid dose).

ORGAN WEIGHTS
All adult animals of the Parental and F1 generations:
organs: brain, pituitary gland, thymus, liver, adrenals, kidneys, prostate, seminal vesicles, testes epididymes or ovaries, uterus
Postmortem examinations (offspring):
SACRIFICE: all unselected offsprings from the P mating and all F1 offspring

GROSS NECROPSY: for all animals of the Parental and F1 generations

HISTOPATHOLOGY
All adult animals from control and high dose groups of the Parental and F1 generations:
organs: ovaries, uterus, cervix, vagina, testes, epididymides, seminal vesicles, prostate, coagulating gland, pituitary gland., significant
abnormalities and liver (potential target organ examined also in low and mid dose).

ORGAN WEIGTHS
All adult animals of the Parental and F1 generations:
organs: brain, pituitary gland, thymus, liver, adrenals, kidneys, prostate, seminal vesicles, testes epididymes or ovaries, uterus
Statistics:
Analyses of bodyweights, food consumption, litter size and weight, offspring landmarks of physical development and absolute organ
weights: homogeneity of variance using Levene's test and one way analysis of variance; Subsequent comparisons between control
and treated groups were performed using Dunnett's T3 Multiple Comparison Method (if unequal variances) or using Dunnett's Multiple
Comparison Method (if equal variances).
Analyses of gestation length, offspring sex ratios, offspring reflexological responses, group mean pre-coital length, sperm concentration,
oocyte counts and organ weights relative to bodyweight: comparison of individual values by Kruskal Wallis non-parametric rank sum test;
if significant differences, pairwise comparison of control values and treated group values by Mann-Whithney "U" test.
Reproductive indices:
Fertility indices:
- male mating index (%)= (number of male mated / number of male paired) x 100
- female mating index (%)= (number of female mated / number of female paired) x 100
- pregnancy index (%)= (number of pregnant/ number of mated females) x 100
- parturition index (%)= (number of females delivering live pups/ number of pregnant females)x 100
Offspring viability indices:
live birth index (%)= (number of pups alive on day 1/ number of pups born) x 100
viability index 1 (%)= (number of pups alive on day 4 before culling/ number of pups alive on day 1) x 100
viability index 2 (%)= (number of pups alive on day 7 before culling/ number of pups alive on day 4 after culling) x 100
viability index 3 (%)= (number of pups alive on day 14/ number of pups alive on day 7) x 100
viability index 4 (%)= (number of pups alive on day 21/ number of pups alive on day 14) x 100
viability index 5 (%)= (number of viable pups at weaning/ number of pups on day 4 after culling) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Mortality (see tables 1 and 2)

P generation
At 11.7 mg/kg, one female was found dead during the gestation phase. No clinical symptom was reported prior to death. Three females
were killed due to clinical findings consistent with possible dystocia.
At 5.85 mg/kg, one female was found dead during the maturation period. Respiratory distress was reported on the day prior to death.
At 0.78 mg/kg, one male was killed during the post mating phase. Respiratory distress was reported on the 3 days prior to death. One
female was killed during gestation. Clinical findings were restricted to the day of death. One female was found dead during the lactation
phase. No clinical symptom was reported prior to death.
In the control group, one male was killed due to twisted paws and bent tail and one female was found dead, with no significant clinical
findings prior to death.

F1 generation
At 11.7 mg/kg, nine males and five females were found dead, plus one male was killed during the maturation phase. There
was no significant clinical history indicative of test material toxicity for these animals.
At 5.85 mg/kg, three males were found dead and one male was killed, without significant clinical findings.
At 0.78 mg/kg, one female was found dead due to dosing trauma.
In control group, two males and two females were either killed or found dead due to dosing trauma during the maturation period.
One female was killed during the gestation/parturition phase, due to evidence of dystocia.

Clinical signs
For both P and F1 generations, there were no treatment or dosage related trends in the incidence or distribution of clinical signs
observed for both males and females. The clinical findings observed were those commonly observed amongst animals for this type of
study.

Body weight (see tables 3 and 4)
In both generations, there were no toxicologically significant effects on body weight gain that could be attributed to treatment.
The only significant differences observed were a slightly reduced bodyweight in the females of the F1 generation at 5.85 mg/kg at the
start of the maturation phase (this difference was likely to have been due to the randomisation of the animals) and a lower bodyweight
in females of P generation at 11.7 and 5.85 mg/kg on day 1 of lactation (not repeated in the F1 generation).

Food consumption
No significant effects of treatment on food consumption were observed

Oestrus cycle
There were no treatment or dosage related trends in the distribution of regular or irregular oestrous cycle lengths for either the P or
F1 generation rats. The incidence of irregular oestrous cycles was within the expected range for this strain of rat.

Fertility (see table 5)
The mating and pregnancy indices for both the P and F1 generations demonstrate that there were no treatment-related effects on
either mating performance or subsequent pregnancy rates.
The majority of mating pairs showed a precoital interval of between 1–4 days for both generations, which is the expected result for this
study type.

Gestation and parturition (see table 6)
There were no significant treatment-related effects on gestation or parturition indices for both the P and F1 generations. The
distribution of gestation lengths showed no significant treatment-related trends.

Litter size and offspring viability (see tables 7 and 8)

P generation
There was a higher than expected incidence of pup deaths prior to Day 4 of lactation, mainly due to a significant number of females
with total litter loss. These litters were distributed across all groups with the highest number seen amongst the high dose females
(11.7 mg/kg). The distribution is such that this is not considered a treatment-related effect.
The majority of the total litter losses occurred between birth and Day 4 of lactation. These litter losses had an effect on the comparison
of mean implantation count to mean number of offspring born, but the results showed there was no treatment-related effect on post
implantation loss values.
At 11.7 mg/kg, group mean litter size was slightly lower than control values, but was not statistically significant and not considered
biologically significant.
The group mean litter size and viability after Day 4 of lactation show no significant effects on offspring survival.

F1 generation
The overall incidence of early pup deaths and total litter loss was considerably lower than that seen in the P generation. There were
no significant inter-group differences in group mean litter size or pup viability throughout the lactation phase. There was no significant
difference in post implantation loss, as identified from comparison of mean implantation count and group mean litter size at birth.

Adult necropsy
With the exception of the necropsy findings associated with decedent animals from either generation, the incidence and type of findings
observed amongst rats (of either sex) were those commonly observed for this strain of rat. There were no treatment-related trends in the
incidence of abnormalities observed.

Organ weight
The only organ weight differences of any consistency seen in one or both sexes for both generations occurred in the liver in animals
treated at 11.7 mg/kg. F1 generation females had a slight increase in absolute liver weight compared to that of the controls (p <0.01).
There was an increase in liver weight, relative to bodyweight for P and F1 generation females compared to controls
(p <0.01 and p <0.001 respectively). There was an increase in liver weight relative to bodyweight for F1 generation males, compared to
controls (p < 0.001).
There were no significant differences in organ weight values in either the intermediate (5.85 mg/kg) or low dose (0.78 mg/kg) groups
when compared to controls for either generation.

Histopathology
P and F1 generations
At 11.7 mg/kg, both male and female adults from both P and F1 generations, had a significant treatment-related incidence of liver
changes. This is consistent with knowledge from other studies that the liver is a potential target organ. A statistically significant incidence (p < 0.001) of periportal hepatocyte vacuolation and enlargement were observed compared to control rats. In addition, P generation
males were observed to have a statistically significant (p < 0.001) incidence of bile duct proliferation compared to controls.
There were no effects observed on any reproductive organ/tissues of either sex, in either generation.
At 5.85 mg/kg, liver changes were observed in both sexes and for both generations. A statistically significant incidence (p < 0.001) of
periportal hepatocyte vacuolation was observed in both sexes and for both generations compared to controls. Periportal hepatocyte
enlargement was observed in males of the P and F1 generations and the incidence was statistically significant (p<0.001) compared to
controls.
There were no effects observed on any reproductive organ/tissues of either sex, in both generations.
At 0.78 mg/kg, no significant findings were observed for target or reproductive organs in animals of either sex or generation.

Sperm assessment
There were no significant treatment-related differences in the proportion of males with each of the sperm motility scores considered for
either generation and no significant differences in the proportion of abnormal sperm observed.

Oocyte assessment
There were no significant treatment-related differences in the overall oocyte numbers observed for either generation. The proportion of
oocyte numbers classified within the different stages of maturation were similar for high dose and control females.
Dose descriptor:
LOAEL
Effect level:
5.85 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Dose descriptor:
NOAEL
Effect level:
0.78 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see below
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Offspring clinical findings
The majority of clinical findings for both the P and F1 generations were associated with adult females that experienced total litter loss.
The most common findings included cold, weak offspring with no milk in their stomach. The offspring were scattered around the home
cage and were often not cleaned of their placenta or amnion. There was evidence of cannibalism, of live as well as dead pups.
These findings were usually noted for the whole litter, mainly in litters totally lost.

Offspring bodyweight and development
There were no significant differences in offspring bodyweight gain throughout lactation for surviving pups in either generation. Group
mean total litter weights were comparable for all dose groups and controls. There were no significant effects seen on inter-group mean
values for intra-litter onset and completion of landmarks of offspring development for either generation.

Offspring refloxological data
There were no significant effects on the group mean percentage of successful intra-litter reflexological responses for offspring from
either generation

Offspring sex ratios and sexual development
There were no effects on intra-litter offspring sex ratios for either generation. There were no effects seen on the time to completion of
external sexual maturity, amongst rats of either sex, for selected F1 animals.

Offspring necropsy
There were no significant treatment-related trends in the proportion of anomalies seen amongst either interim deaths or terminal kill
offspring. The majority of the findings amongst interim death offspring were associated with the adult females with total litter loss,
particularly in relation to no milk in the stomach of these pups and to evidence of cannibalism.

Sperm assessment
There were no significant treatment-related differences in the proportion of males with each of the sperm motility scores considered for
either generation and no significant differences in the proportion of abnormal sperm observed.

Oocyte assessment
There were no significant treatment-related differences in the overall oocyte numbers observed for either generation. The proportion of
oocyte numbers classified within the different stages of maturation were similar for high dose and control females.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
5.85 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.78 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Dose descriptor:
NOAEL
Remarks:
(highest dose tested)
Generation:
F2
Effect level:
11.7 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Sex:
male/female
Basis for effect level:
other: overall effects
Reproductive effects observed:
not specified

Table 1: Results of mortality (males)

Dose [mg/kg bw/d]

Parental

F1generation

Active substance

Main ingredient

Killed in extremis

Found dead

Killedin extremis

Found dead

0

0

1

0

1

1

1.0

0.78

1

0

1

1

7.5

5.85

0

0

1

3

15.0

11.7

0

0

1

9

 

Table 2: Results of mortality (females)

Dose [mg/kg bw/d]

Parental

F1generation

Active substance

Main ingredient

Killed in extremis

Found dead

Killedin extremis

Found dead

0

0

0

1

1

2

1.0

0.78

1

1

0

1

7.5

5.85

0

1

0

0

15.0

11.7

3

1

0

5

Table 3: Results of female body weight prior to pairing (mean in g)

Week of dosing

Parental generation

(dose as mg/kg bw/d as main ingredient)

F1generation

(dose as mg/kg bw/d as main ingredient)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.85)

Group 4

(11.7)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.86)

Group 4

(11.7)

1

160

158

158

157

80

81

71***

79

2

181

183

183

180

121

121

110***

117

3

207

206

205

205

159

159

149*

151

4

227

225

227

224

186

186

176*

181

5

242

240

239

238

205

211

198

203

6

256

254

251

252

228

231

219

223

7

267

264

263

266

242

248

238

239

8

276

273

272

273

256

263

250

252

9

283

280

279

277

267

274

261

265

10

290

287

286

283

275

284

269

273

11

295

292

292

291

284

291

278

282

12

 

 

 

 

292

300

286

290

* :        significantly different from control value (p < 0,05)

*** :     significantly different from control value (p < 0,001)

Table 4: Results of female body weight during lactation (mean in g.)

Daypost coitum

Parental generation

(dose as mg/kg bw/d as main ingredient)

F1generation

(dose as mg/kg bw/d as main ingredient)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.85)

Group 4

(11.7)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.86)

Group 4

(11.7)

1

299

287

279*

281*

303

306

298

302

4

320

317

307

310

329

336

323

327

7

334

332

321

322

340

350

334

339

14

359

359

356

350

363

377

361

366

21

359

362

357

345

364

378

358

362

* :        significantly different from control value (p < 0,05)

Table 5: Summary results of mating performance and fertility


Parental generation

(dose as mg/kg as main ingredient)

F1generation

(dose as mg/kg as main ingredient)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.85)

Group 4

(11.7)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.86)

Group 4

(11.7)

males paired

 

31

 

32

 

31

 

32

 

30

 

31

 

28

 

22

females paired

 

32

 

32

 

31

 

32

 

30

 

31

 

32

 

27

females mated

 

32

 

32

 

31

 

32

 

30

 

31

 

32

 

27

females pregnant

 

31

 

32

 

31

 

29A

 

25

 

25

 

31

 

25

Male mating index (%)

 

100

 

100

 

100

 

100

 

100

 

100

 

100

 

100

Pregnant index (%)

 

96.9

 

100

 

100

 

96.7

 

83.3

 

80.6

 

96.9

 

92.6

A= for calculation purposes 2 females were excluded due to insufficient data recorded to establish pregnancy status

Table 6: Summary results of gestation indices

 

Group

(dose as mg/kg main ingredient)

 

No. females mated

 

Gestation Length (days)

 

No.

pregnant

No. females with live

born offspring

 

22

 

22.5

 

23

 

23.5

 

24

 

25

Parental generation

1 (control)

32

8

6

15

0

0

1

31(30)

30

2 (0.78)

32

5

8

17

0

1

0

32(31)

31

3 (5.85)

31

9

5

17

0

0

0

31

31

4 (11.7)

32

7

3

16

2

0

0

29A(27)

27

F1generation

1 (control)

30

8

7

9

0

0

 

25(24)

24

2 (0.78)

31

5

5

14

1

0

 

25

25

3 (5.85)

32

9

9

12

1

0

 

31

31

4 (11.7)

27

6

5

12

1

1

 

25

25

A= for calculation purposes 2 females were excluded due to insufficient data recorded to establish pregnancy status

(  ) = value used for calculation of parturition index, excludes females with insufficient evidence of completed parturition.

Table 7: Summary results of live birth and viability indices

 

P-F1generation

(dose as mg/kg as main ingredient)

F1– F2generation

(dose as mg/kg as main ingredient)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.85)

Group 4

(11.7)

Group 1

(control)

Group 2

(0.78)

Group 3

(5.86)

Group 4

(11.7)

Live Birth Index

(%)

 

91.3

 

95.3

 

90.5

 

88.4

 

98.2

 

93.1

 

95.4

 

93.4

Viability Index (%)

Group 1

(control)

Group 2

(1 mg/kg)

Group 3

(7.5 mg/kg)

Group 4

(15 mg/kg)

Group 1

(control)

Group 2

(1 mg/kg)

Group 3

(7.5 mg/kg)

Group 4

(15 mg/kg)

1

74.8

84.6

77.1

70.2

94.4

87.3

98.5

88.9

2

100

99.6

100

100

99.4

100

99.6

100

3

98.9

98.2

100

99.3

98.9

100

99.6

100

4

100

99.5

99.5

100

100

100

100

100

5

98.9

97.3

99.5

99.3

98.3

100

99.2

100

Conclusions:
Under the test conditions of this study, the NOAEL for both males and females of P and F1 generation was 0.78 mg/kg bw/day (expressed as main ingredient) or 1 mg/kg bw/day (expressed as active substance) based on liver effects. The LOAEL for both males and females of P and F1 generation was 5.85 mg/kg bw/day (expressed as main ingredient) or 7.5 mg/kg bw/day (expressed as active substance).Therefore, THPS 100% or 75% were considered as not reprotoxic and not classified according to EU criteria.
Executive summary:

THPS (78.0% THPS main ingredient) was tested in accordance with US EPA Pesticide Assessment Guidelines, Subdivision F, § 83-4 and OECD Guideline 416 and in compliance with GLP.

Thirty-two Sprague-Dawley rats/ sex/ dose were given test substance at 1,7.5 and 15 mg/kg bw/d as active substance (0.78, 5.85 and 11.8 mg/kg bw/d as main ingredient) by gavage. Both male and female parental animals and their descendants of each sex, the F1 generation, were dosed. Parental males and females were dosed for 76 days prior to pairing and for up to 13 days during mating. The test material was then administered only to females during gestation and lactation phases until Day 21 post partum. Selected F1 animals were dosed from Day 21 post partum for 82 days prior to pairing.The test material was then administered only to F1 females during gestation and lactation phases until Day 21 post partum. 

The dosing of test material to two successive generations of the rat at dose levels of 5.85 and 11.7 mg/kg resulted in morphological changes in the liver of both male and female adults of both P and F1 generations. The No Effect Level for effects on the liver was 0.78 mg/kg bw/d.

There were no effects observed at any dose level on reproduction during the course of the study.

During the study a number of mortalities were observed, the highest incidence was seen amongst high dose F1 rats. The majority of these deaths occurred during the first two weeks after initiation of dosing, and on post mortem examination pneumonitis was diagnosed. The deaths were therefore attributable to dosing trauma and did not represent any evidence of systemic toxicity.

Significant increase of periportal hepatocyte vacuolation and enlargement were observed in the 5.85 and 11.8 mg/kg bw/d treated animals when compared to control rats.

The reproduction phases of the study, particularly in the P generation, showed a higher than expected incidence of early pup deaths, mainly due to a significant number of females with total litter loss prior to Day 4 of lactation.

This random pattern of total litter losses across all groups is consistent with a pattern of events experienced by research laboratories throughout UK with this particular strain of rat and which has been documented in a publication [Teratology, 1999: 59 (6) p410]. The finding is related to the dietary intake and is not the result of test material administration. Substituting the diet for one used by the breeders eliminated the problem in subsequent studies in all laboratories.

The F1 generation showed lower levels of total litter loss consistent with the P generation rats most sensitive to the subtle dietary insufficiency failing to produce any progeny.

Clinical signs and necropsy findings for pups dying during the lactation phase (pups with no milk in the stomach, cold, weak, scattered, sometimes cannibalised) are indicative that poor maternal care due to maternal dietary insufficiency not treatment-related toxicity was the probable cause of the high pup mortality.

At 11.7 mg/kg, there were no significant findings for effects on reproductive performance in either generation. Histopathology, semen analysis and oocyte evaluation failed to show any significant treatment-related effects on the reproductive organs of either generation.

At 5.85 mg/kg, there were no significant effects on reproductive performance and no effects on the reproductive organs for either generation.

At 0.78 mg/kg, there were no effects on reproductive performance or on the reproductive organs for either generation.

Based on these results:

- The NOAEL for both males and females of P and F1 generation was 0.78 mg/kg bw/day (expressed as main ingredient) or 1 mg/kg bw/day (expressed as active substance) based on liver effects.

- The LOAEL for both males and females of P and F1 generation was 5.85 mg/kg bw/day (expressed as main ingredient) or 7.5 mg/kg bw/day (expressed as active substance).

In conclusion, THPS 100% or 75% were considered as not reprotoxic and not classified according to EU criteria.

This reproductive toxicity study is classified as acceptable. It satisfies the guideline requirement for a 2 generation reproductive toxicity study in the rat.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION:
see attached justification document.
Reason / purpose:
read-across source
Key result
Dose descriptor:
LOAEL
Effect level:
5.85 mg/kg bw/day
Based on:
other: act.ingr/THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Key result
Dose descriptor:
NOAEL
Effect level:
0.78 mg/kg bw/day
Based on:
other: act.ingr/THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
5.85 mg/kg bw/day
Based on:
other: act.ingr/THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.78 mg/kg bw/day
Based on:
other: act.ingr/THPS
Sex:
male/female
Basis for effect level:
other: histopathological liver effects
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
11.7 mg/kg bw/day
Based on:
other: act.ingr/THPS
Sex:
male/female
Basis for effect level:
other: Overall effects
Key result
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD 416, Kr.2, GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

See attached justification document (7.8.1/Tox to repro (RA from data source).

Effects on developmental toxicity

Description of key information
Oral developmental toxicity study in rabbits: LOEL (maternal): 6.2 mg AI/kg/day bw; NOEL (development): 25 mg AI/kg bw/day (OECD 414, GLP, Rel. 1).
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

THPC-urea-amine (Perform STi) was administered to 24 pregnant female NZW rabbits per group at 0, 10, 40 and 120 mg/kg bw/day as test substance (approximately 6.2, 25 and 75 mg AI/kg bw/day)over Days 6 to 28 of gestation and terminated on Day 29.

Severe maternal toxicity was evident at the high dose level of 75 mg AI/kg bw/day and included marked body weight loss and marked food consumption reduction, observed in almost all animals in this group, and reduced/altered faecal output, observed in all high dose animals. Clinical signs including laboured breathing, subdued behaviour and signs of aborting litters required the premature sacrifice of 6/24 animals in this group. Liver weights were increased, and necropsy findings included an increased incidence of pale livers and prominent lobulation. Histopathology findings included periportal hepatocyte vacuolation observed in all animals in this group, with centrilobular vacuolation, hepatocyte necrosis and periportal hepatocyte hypertrophy also observed in premature decedents. These liver findings were preceded by an increase in the levels of AST,ALT, LDH and a reduction in ALP on GD 13 and an increase in ALT, Cholesterol, Triglycerides, GGT, and reduction in Haemoglobin and Red Blood Cell counts on GD 29. These clinical pathology effects are consistent with changes that would be associated with the liver findings.

Adverse effects on development were reported only in the high dose group receiving 75 mg AI/kg bw/day. At this dose level there was an increase in the number of dead implants, mainly early embryonic deaths. Mean foetal weights were lower than Control, and there was a marked increase in the incidences of foetal abnormalities and variants. The principal findings were eye (eye/lens reduced, retinal folds, lenticular/corneal opacities) and hindlimb/digit (absent/fused/poorly defined bones) abnormalities, with many foetuses having both eye and hindlimb/digit findings; haemorrhages in the orbital socket (usually in foetuses with eye effects); foetuses with incisors not erupted; reduced state of skeletal ossification, probably associated with the small size of the foetuses; greater incidences of foetuses with bilateral caudal displacement of the pelvic girdle and of foetuses with supernumerary ribs: foetuses with pelvic girdle displacement generally had 13 complete ribs. It was considered not clear if the above foetal findings were directly related to treatment or secondary to the severe maternal effects.

At 25 mg AI/kg/day, there was an increased incidence of females with reduced faecal output. The only necropsy findings that appeared to be associated with treatment were pale livers in 4 females; histopathology indicated 6 females with periportal hepatocyte vacuolation, but to a lesser degree than for the females at 75 mg AI/kg/day.

At 6.2 mg AI/kg/day, two animals had pale liver but there were no histological changes and no effects on liver weights at this level; it is not clear if these liver findings were incidental or related to treatment with Perform STi.

The type and distribution of foetal visceral and skeletal abnormalities at 6.2 and 25 mg AI/kg/day were not considered to be associated with treatment.

In conclusion, no clear maternal no observed effect level (NOEL) could be established. There was a clear foetal NOEL of 25 mg AI/kg/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Key study

Justification for classification or non-classification

Fertility:

By analogy with bis[tetrakis(hydroxymethyl) phosphonium] sulphate”, CAS Number: 55566-30-8 at 78% (OECD 416, GLP, Kr.2), the test substance is not considered as reprotoxic for fertility and not classified according to EU criteria.

Development:

According toDirective (EC) No. 1272/2008:

3.7.2.4.4: Maternal mortality: an increased incidence of mortality among the treated dams over the controls shall be considered evidence of maternal toxicity if the increase occurs in a dose-related manner and can be attributed to the systemic toxicity of the test material.Maternal mortality greater than 10 % is considered excessive and the data for that dose level shall not normally be considered for further evaluation. […]

6/24 pregnant females at 75 mg AI/kg bw/day were sacrificed prematurely due to severe clinical signs including laboured breathing, subdued behaviour and signs of aborting litters in the later part of the exposure period. These effects were considered test substance-related. Effectively mortality was 25%. For surviving high dose dams, mean bodyweight gain was approximately 50% that of controls, with a mean bodyweight loss during GD6-12. Mean food consumption was significantly reduced, with overall consumption 54% that of control animals and approximately 30% that of controls during the period GD7-19.

The high level of maternal mortality at75 mg AI/kg bw/day, in combination with the severity of the effects on surviving animals indicates that it is reasonable to conclude that the developmental effects reported (mainly eye and hindlimb abnormalities, small size, reduced/delayed ossification) the foetal data for this dose level should not be considered for evaluation and classification.

There were no foetal visceral or skeletal abnormalities considered related to treatment at either 6.2 or 25 mg AI/kg bw/day, both doses causing maternal toxicity.

According to the above criteria of Directive (EC) No. 1272/2008, it could be concluded that no classification for developmental toxicity is required.

However, there is evidence of similar eye and limb malformations from OECD 414 studies on other THP+ substances in the presence of maternal toxicity. A direct substance-related effect cannot be excluded.

Taking a weight of evidence approach, it is therefore concluded that THPC-urea-amine should be classified for reproductive toxicity as Category 2 Suspected human reproductive toxicant (H361d – Suspected of damaging the unborn child), according to CLP regulation 1272/2008.