Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
other:
Adequacy of study:
supporting study
Study period:
Not applicable
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A detailed review of the available study data for developmental toxicity comparing the observed effects against the relevant classification criteria in accordance with Regulation EC 1272/2008 (CLP) (Annex II points 3.7.2.3 and 3.7.2.4) considering the effect of the observed maternal toxicity on the pregnancy outcomes of the test animals. The review of the information was presented as a formal report.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
An expert assessment of the appropriate classification for developmental toxicity of MBTC under CLP, with particular respect to the results of the key rabbit study submitted to support the registration. A detailed review of individual pregnancy outcomes was performed by an expert toxicologist and were correlated with the maternal findings in order to assess the influence of maternal toxicity to pregnancy outcome.
GLP compliance:
no
Remarks:
not appropriate
Type of method:
other: Expert review of the available study and literature data

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: rat and rabbit

Results and discussion

Any other information on results incl. tables

Key Study– Rabbit Study

Pregnancy outcomesin the main study can be summarized as:

Table 1: Key pregnancy outcomes with individual animal identities

Dose, mg/kg bw/day

0

30

60

120

Number of females

25

25

25

25

Non-pregnant

0

1

0

1

Died or euthanized

0

1 (247)

1 (252)

1 (282)

Abortion

0

0

1 (257)

2 (278, 281)

Early delivery

0

0

0

1 (288)

Litters entirely resorbed in utero

0

0

0

2 (280, 292)

Viable litters at term

25

24

23

18

Individual animal ID numbers of affected rabbits given in parentheses

At the top dose, fewer litters successfully completed pregnancy compared to controls. Of these, one female was non-pregnant, and one female (ID number 282) died with cause of death attributed to a dosing injury. Neither of these can be attributed to the test material.

For the remaining affected animals, individual bodyweight and food consumption data are presented in Table 5. Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption, and a clear treatment-related effect is evident on both food consumption and group mean bodyweight at the top dose level. In Table 5, individual values are indicated as showing a “clear deficit” when there is any weight loss from the previously recorded bodyweight; or a food consumption value less than half of the mean value of the top dose group (i.e. these animals being particularly affected within the group).

Three animals aborted (one at 60 mg/kg bw/day and two at the top dose), and one (at the top dose) delivered prematurely. The study report specifically attributes these to maternal toxicity (low weight gain, prolonged inappetance and clinical signs). Table 5 demonstrates that the affected animals showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group. The current report concurs that these failures of pregnancy can be directly attributed to the maternal effects; maternal well-being was insufficient to maintain pregnancy.

One possible divergence of opinion might be the animal that delivered early. The author could not support premature delivery as a recognized consequence of maternal toxicity, although this animal showed marked toxicity in this study. The author noted that premature delivery in teratogenicity studies can be an occasional technical flaw resulting from mis-timing the day of conception (the rabbits in this study were naturally mated, not artificially inseminated). The litter of 7 kits appears otherwise normal from the report and accounted for all implantation sites; no effect on development can be inferred. Early delivery, even if plausibly treatment-related, however is insufficient evidence for classification in this study since it is confined to a single animal.

Two animals (both in the top dose group) showed total litter resorptions in-utero. These were reported as “early” resorptions. Once again, as can be seen from Table 5 each of these animals showed prolonged periods of markedly deficient food consumption and weight gain. It is not possible to establish a precise timepoint at which each pregnancy failed. However, it is consistent with the data to hypothesise that each pregnancy failed within the period during which marked maternal toxicity was expressed.

The magnitude of the food consumption deficit is considerable. Assuming a normal control food intake at 140 g/animal/day, a 9-day period consuming 5 g/day or less (which occurred in 6 of the 7 animals) represents a deficit of approximately 1.2 kg of food; equivalent to nearly half of each animals’ bodyweight.

Increased post-implantation losswas attributable to the two litters showing complete resorption, in turn a consequence of marked maternal toxicity. When these two values are excluded from the calculation of the group mean, there is no clear increase:

Table 2: Adjusted post-implantation loss, rabbit study

Dose, mg/kg bw/day

0

30

60

120

Post implantation loss, % (report p64)

2.09

4.61

3.06

15.32

Excluding 2 litters with all resorptions:

 

 

 

6.6

The reported increases in total and early resorptions are again attributable to these two litters, both of which were reported as early resorptions. A post-implantation loss rate of 4.6% is reported at 30 mg/kg bw/day and considered a no-effect level; the high dose rate at 6.6% is not considered biologically different. (No statistical analysis of these data is offered either in the study report or in this evaluation).

Lower foetal weightswere significant at the high dose only.These however mirrored maternal weight change, and can be attributed to the maternal weight effect.

 

Table 3: Association of maternal and foetal weight effects, rabbit study

Dose, mg/kg bw/day

0

30

60

120

Final maternal bodyweight, mean (kg)

3.6

3.6

3.6

3.4**

Maternal adjusted weight gain (kg)

0.04

0.03

0.01

-0.13**

Foetal bodyweight, mean (g)

40.0

40.0

37.9

35.6**

Level of statistical significance: ** p > 0.01 in comparison with control

Table 5: Comparison of rabbit food consumption and weight gain in unsuccessful pregnancies – Key rabbit study

 

 

Study Period (days)

 

 

0

1

3

6

9

12

15

18

21

24

27

29

Control group mean

BW

3.13

3.19

3.25

3. 31

3.36

3.40

3.50

3.52

3.55

3.60

3.63

3.65

 

FC

 

52

151

156

150

147

133

143

142

112

93

88

High dose group mean

BW

3.13

3.15

3.17

3.15*

3.16**

3.18**

3.26**

3.26**

3.31**

3.36**

3.40*

3.42*

 

FC

 

50

109**

91**

72**

67**

64**

71**

88**

91

77

59

 

257 - aborted

BW

3.25

3.28

3.29

3.38

3.32

3.38

3.34

3.31

3.23

3.22

3.24

NDC

 

FC

 

55.0

125.5

140.7

77.0

84.7

10.0

3.7

11.7

25.3

34.0

NDC

278 - aborted

BW

3.47

3.58

3.51

3.38

3.27

3.26

3.28

3.34

3.33

3.25

NDC

 

 

FC

 

50.0

134.0

40.7

0.7

6.0

0.7

0.3

0.3

0.7

0.0

NDC

280 – all resorbed

BW

3.24

3.24

3.27

3.17

3.14

2.98

2.97

2.88

2.76

2.99

3.11

3.08

 

FC

 

36.0

114.0

60.7

0.3

5.3

1.0

2.0

2.3

127.0

155.0

97.5

281 - aborted

BW

3.31

3.39

3.33

3.21

3.15

3.11

3.15

3.00

3.25

NDC

 

 

 

FC

 

47.0

35.0

18.7

2.51

3.51

0.01

0.0

33.7

0.0

NDC

 

288 – premature

BW

2.58

2.65

2.63

2.73

2.77

2.74

2.79

2.73

2.74

2.74

2.83

NDC

 

FC

 

59.0

103.5

104.3

84.0

71.3

21.3

2.7

1.7

1.3

4.0

NDC

292 – all resorbed

BW

3.16

3.10

3.17

3.15

3.06

2.96

3.18

3.35

3.35

3.41

3.50

3.52

 

FC

 

30.0

105.5

66.7

0.0

16.0

100.0

107.0

108.3

123.0

169.3

152.5

BW: bodyweight (kg); FC: food consumption (g/animal/day during the period between bodyweight measurement). Values in bold show clear deficits from normal.

NDC: no data collected. Level of statistical significance: * p >0.05, ** p> 0.01 in comparison with control.

 

Key Study - Rat Study

The only reproduction-related outcome reported as a consequence of treatment was marginally lower foetal weights, seen only at a dose level where maternal food consumption and bodyweight gain were impaired:

Table 6: Association of maternal and foetal weight effects, rat study

Dose, mg/kg bw/day

0

100

300

1000

Food consumption/ g/rat/day GD 0-20

19.5

18.9

19.7

17.7**

Final maternal bodyweight, mean (g)

371

364

378

346*

Maternal adjusted weight gain (g)

83

84

93

66**

Foetal bodyweight, mean (g)

4.19

4.09

4.15

3.83**

Level of statistical significance: * p>0.05, ** p>0.01 in comparison with control

 

Lower foetal weights in the presence of impaired maternal weight gain tends to be a finding of low concern, and is generally accepted as a direct consequence of the maternal toxicity.

 

Other relevant literature:

Nakamura et al (1992; in Japanese) demonstrated the presence of n-butyltins (mono- and di-butyl) in the foetus and placenta of rats following oral administration of di-butyltin diacetate, which was seen to be teratogenic. This observation supports consideration of the foetus as a target for the observed pregnancy failures seen in the key rabbit study. Although not excluding the possibility of a relevant local effect of butyltin trichloride in the rabbit, a local effect appears unlikely on balance of evidence. Systemic and foetal exposure is in any case the default assumption in evaluating developmental toxicity.

Noda et al (1992) reported the comparative teratogenicity of di-butyltin diacetate with n-butyltin trichloride (MBTC). Di-butyltin diacetate was teratogenic; whereas n-butyltin trichloride (MBTC, at doses up to 400 mg/kg bw/day) showed no teratogenicity in this study. This observation both supports the findings of the key rat study with n-butyltin dichloride (no teratogenic effect in rat, at a higher dose) and mitigates concern arising from observation of developmental toxicity with di-butyltin diacetate in Nakamura et al (1992). At doses up to 400 mg/kg bw/day of n-butyltin trichloride, Noda (1992) demonstrated no maternal or foetal toxicity of MBTC in rats, with the exception of reduced thymic weight in maternal animals. This study is Noda (1992b), mentioned below.

ASTDR catalogue several studies of butyltin chlorides (for citations see ASTDR). These include:

Ema et al (1991c) reporting increased numbers of resorptions, dead foetuses and post-implantation loss with dibutyltin dichloride in the absence of maternal toxicity, but at doses (ca 7.5 mg/kg bw/day) much lower than MBTC as tested in the key study in rats. A second study (Farr et al, 2001) reported maternal toxicity at 10 mg/kg bw/day without foetal effect, therefore conflicting with Ema. Noda (1992b; ASTDR p144) is cited to show teratogenicity with dibutyltin dichloride at 10 mg/kg bw/day, although this may be an error: this reference reports the comparative study of d-butyltin diacetate with dibutyltin dichloride, and no teratogenicity was seen with dibutyltin dichloride. Ema et al (1992) reported a sensitive window for dibutyltin dichloride on post-implantation loss at days 7-9 of pregnancy, but no discussion of any concurrent maternal toxicity is given. These doses are substantially below the 1000 mg/kg bw/day MBTC tested by the key rat study, and the two tin compounds (dibutyltin dichloride and MBTC) do not appear toxicologically comparable. Harazono and Ema (2003) appear to show pre-and post-implantation losses with dibutyltin dichloride as preventable by progesterone administration. This speaks to a specific effect on pregnancy, but these pre-implantation and early post-implantation losses were not seen in the key study in rats; and dibutyltin dichloride is not the same as MBTC. Post-implantation losses in the key rabbit study of MBTC might be relevant, but occurred in the form of 2 complete litter losses; it is not possible to determine from the ASTDR document if the same pattern is seen.

Further work by Ema and colleagues (1991c, 1997b) showed malformations (particularly cleft palate) with tributyltin chloride. Maternal toxicity is however not discussed. Once again, doses were markedly lower (ca. 10 mg/kg bw/day) than the 1000 mg/kg bw/day MBTC as tested in the definitive, guideline rat study (provided as the key); suggesting again that the toxicities of MBTC and tributyltin chloride are dissimilar. In the definitive GLP and guideline study in rats, provided as the key study, MBTC showed no evidence for malformation.

ASTDR concludes a number of “reliable” NOAELs from these publications, suggesting the quality of the literature has been assessed. The relevant literature does not appear to include studies of the developmental toxicity of butyltins in rabbits.

In summary of the literature: data on analogous butyltin compounds shows some evidence for developmental toxicity in rats, but literature on rabbits does not appear to be available. The clearest evidence for at least two butyltins (dibutyltin diacetate and tributyltin chloride) was for malformation, which clearly did not occur with MBTC (based on the key rat and rabbit studies conducted in accordance with OECD 414). Pre-implantation and early post-implantation losses were seen in a number of the literature studies but (where discussed) appear most often to be at levels of significant maternal toxicity. One report does suggest the early implantation losses in rats to be prevented by progesterone treatment, thus suggesting a specific and direct effect on pregnancy for which classification would be appropriate. The work does not appear to have been corroborated by a second institution. Extrapolation of this rat finding with dibutyltin dichloride to rabbits with MBTC may be tenuous particularly with respect to the specific endocrinology involved. Post-implantation loss was not seen in rats with MBTC (key rat study). ASTDR discusses endocrinology of organic tin compounds without support for effects on progesterone; it is unclear if progesterone was investigated in any of the studies cited. The chemical analogies are not close, and sensitivity of the maternal animal to the other butyltins appears markedly greater than seen for MBTC. Literature on the butyltins does not show evidence for abortion and early delivery as seen in the key rabbit study; but the literature primarily describes studies in rats with none in rabbits. It must be noted that an accumulation of study reports such as ASTDR is likely to contain some findings that may arise as a function of the quantity of testing, and that specificity and reproducibility become important considerations; classification should not be a function of the quantity of testing.

On balance, the author assesses the studies reported in literature to give inadequate evidence that the findings in the key rabbit study represent a specific effect on pregnancy or the developing foetus such as to justify classification.

Applicant's summary and conclusion

Conclusions:
The CLP Regulation recognizes that certain effects on pregnancy might arise as a result of maternal toxicity, and as such might not be appropriate for classification. For classification to be appropriate “effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects”. Examples cited are: “depressed foetal weight, retarded ossification, and possibly resorptions and certain malformations in some strains of certain species.”
In the key rabbit study, all treatment-related effects on pregnancy fall within these categories, with the exception of abortion. Abortion is however generally recognized as an effect that may be maternal, and is attributed by the Study Director in the study report to maternal toxicity. The author of the review agreed with this opinion.
The maternal effects seen in the affected rabbits are typical of the substance-related toxicity evident in this study. The particular does showing individual adverse pregnancy outcomes are shown to be particularly severely affected, even within the group. Further, the nature of pregnancy loss – affecting the entire litter, not individual pups – is suggestive of an effect on the mother, not on the foetus.
In the key rat study, the only treatment-related effect on development was lower foetal weight in the presence of maternal weight gain impairment. This finding is cited in the wording of the CLP Directive: “Classification is not necessarily the outcome in the case of minor developmental changes, when there is only a small reduction in .. foetal weight .. when seen in association with maternal toxicity.”
In conclusion, there is convincing evidence that all effects on pregnancy in these studies are secondary and directly attributable to maternal toxicity.
No classification for developmental toxicity is appropriate under CLP
Executive summary:

An expert assessment of the appropriate classification for developmental toxicity of MBTC under CLP, with particular respect to the results of the key rabbit study submitted to support the registration. A detailed review of individual pregnancy outcomes was performed by an expert toxicologist and were correlated with the maternal findings in order to assess the influence of maternal toxicity to pregnancy outcome.

The CLP Regulation recognizes that certain effects on pregnancy might arise as a result of maternal toxicity, and as such might not be appropriate for classification. For classification to be appropriate “effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects”. Examples cited are: “depressed foetal weight, retarded ossification, and possibly resorptions and certain malformations in some strains of certain species.”

In the key rabbit study, all treatment-related effects on pregnancy fall within these categories, with the exception of abortion. Abortion is however generally recognized as an effect that may be maternal, and is attributed by the Study Director in the study report to maternal toxicity. The author of the review agreed with this opinion.

The maternal effects seen in the affected rabbits are typical of the substance-related toxicity evident in this study. The particular does showing individual adverse pregnancy outcomes are shown to be particularly severely affected, even within the group. Further, the nature of pregnancy loss – affecting the entire litter, not individual pups – is suggestive of an effect on the mother, not on the foetus.

In the key rat study, the only treatment-related effect on development was lower foetal weight in the presence of maternal weight gain impairment. This finding is cited in the wording of the CLP Directive: “Classification is not necessarily the outcome in the case of minor developmental changes, when there is only a small reduction in .. foetal weight .. when seen in association with maternal toxicity.”

In conclusion, there is convincing evidence that all effects on pregnancy in these studies are secondary and directly attributable to maternal toxicity.

No classification for developmental toxicity is appropriate under CLP