Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study planned
Study period:
The study will be started after approval by ECHA
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS : Toxicity to reproduction
N-methylaniline (NMA) registration dossier was first submitted by the company Rompetrol at > 1000 tpy. However, due to a lack of data, the registration dossier was rejected by ECHA. Indeed, two tests, submitted each to a testing proposal, were required by the Agency for the completeness of the dossier: a 90-day repeated dose toxicity by oral route (OECD 408) and a two-generation reproductive toxicity study (OECD 416) or extended one-generation reproductive study (OECD 443).
The toxicity to reproduction test is only performed if the 90-day repeated dose toxicity study by oral route indicates adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.
Rompetrol decided not to perform these tests because they were too expensive. Rompetrol finally chose to cease the manufacturing of NMA. The registration was stopped and no longer required by ECHA.

More recently, the company Chimec decided to register NMA at the tonnage 100-1000 tpy. The role of Lead Registrant was transferred from Rompetrol to Chimec, which bought the data of NMA registration dossier to Rompetrol. ECHA lowered the Joint submission tonnage band to allow Chimec submitting its dossier. Afterwards, when Chimec submitted the NMA registration dossier, the manual technical completeness check was invalidated by ECHA.

ECHA asked Chimec to complete the justifications of two testing proposals in the IUCLID dossier, i.e. Repeated dose toxicity by dermal route and Toxicity to reproduction. The considerations for why the adaptation possibilities provided by the REACH Regulation cannot be used to address the information requirement were considered incomplete by the Agency.


NON-CONFIDENTIAL NAME OF SUBSTANCE: N-methylaniline

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- Available GLP studies: No available GLP study for the endpoint considered were found for NMA.
- Available non-GLP studies:
3 studies were found concerning NMA reprotoxicity [1, 4, and 16].
The first study (found on TOXNET and PubMed database) [16] is a developmental study. However, this study does not fulfil the information requirement of ECHA as this is only a developmental study and not a longer study such as an extended one-generation or a two-generation reproductive toxicity study.
The second and third studies (found on PubMed database) [1, 4] concern the aquatic species reprotoxicity. These studies are therefore not relevant for the reproduction endpoint as defined in the context part of the present document.

- Historical human data:
No human information has been found for NMA (epidemiology studies on exposed populations/case reports/controlled studies/information from occupational surveillance/postmarketing surveillance data/meta-analysis).

- (Q)SAR:
“There are a large number of potential targets/mechanisms associated with reproductive toxicity which, on the basis of current knowledge, cannot normally be adequately covered by a battery of QSAR models.”
“QSAR approaches are currently not well fitted-for-purpose for reproductive toxicity and consequently no firm recommendations can be made concerning their routine use in a testing strategy in this area.”
 The QSAR approach is not relevant here for this endpoint

- In vitro methods:
“The design of alternatives to in vivo testing for reproductive toxicity is especially challenging in view of the complexity of the reproductive process and large number of potential targets/mechanisms associated with this broad area of toxicity. In addition, many in vitro approaches do not include elements of biotransformation which, in addition, may differ depending on organ.”
“Currently there are only three officially adopted EU test methods or OECD test guidelines for in vitro tests of relevance to modes of action for reproductive toxicity: two measuring oestrogenicity (OECD TG 455 and OECD TG 457) and the other measuring steroidogenesis (EU B.57, OECD TG 456).”
 The available in vitro test methods are not relevant for the reproductive endpoint here.

- Weight of evidence:
NMA is readily absorbed by all three exposure routes (inhalation, dermal and oral) (IUCLID dossier of NMA).
 Therefore, the reproductive study requested by ECHA is relevant for oral route of exposure, the testing is therefore technically possible [2].

- Grouping and read-across:
Data covering NMA are sparse. Based on its chemical structure, the effects may be expected to be in-between those of N,N-dimethylaniline and aniline.

Aniline
- A study was available on aniline hydrochloride [13].
This one-generation reproductive toxicity study aims to investigate teratologic and postnatal effects in rats administered aniline hydrochloride at doses of 10, 30 or 100 mg/kg/d.
“On gestation day 20, dams exhibited characteristic signs of aniline HCl toxicity, i.e., methemoglobinemia, increased relative spleen weight, decreased red blood cell count and hematological changes indicative of increased hematopoietic activity. Fetuses from aniline-treated dams exhibited increased relative liver weight and enhanced hematopoietic activity, but no evidence of an embryolethal or teratogenic effect was observed. High-dose dams exhibited mild methemoglobinemia, increased relative spleen weight, and increased red blood cell count size at termination on pnd 30. Postnatal signs of toxicity in litters from aniline-treated dams (i.e., decreased body weight, elevated relative liver weight, and elevated relative spleen weight) were transient, and no evidence of toxicity was observed in pups surviving to PND 60.Hence, the NOAEL for the F1 generation and LOAEL of the pregnant females were considered to be 10 mg/kg/day when exposed to aniline HCl by oral gavage.”
 This study does not deal with the fertility endpoint, which is necessary to fill the information gap of the IUCLID dossier.

- The SCOEL [15] mentions that aniline can cross the placenta and induce foetal methaemoglobinaemia; a similar property can be expected for N-methylaniline.
 No other information was available. This statement is a kind of alert but is not enough to fulfil the endpoint of NMA registration dossier.

- The registration dossier of N,N-dimethylaniline [12] mentions an one-generation reproductive study in mice where the maternal as well as the developmental effects of aniline given orally at 560 mg/kg/d were assessed. Treatment with aniline had no apparent effect on the number of live litters produced; however offspring viability through the first three postpartum days was significantly lower than that of the control group. In addition, reductions in birth weight and weight gain were seen in the aniline-treated litters. No apparent effect on reproductive outcome were noted
 This study shows that aniline doesn’t lead to maternal reproductive performance outcome, but induces toxicity towards the litters.

N,N-dimethylaniline
- The registration dossier of N,N-dimethylaniline [12] mentions an one-generation reproductive study in mice where the maternal as well as the developmental effects of N,N-dimethylaniline given orally at 365 mg/kg/d were assessed. No adverse effects on survival or weight gain of the dams, or birth weight, weight gain or viability of the F1 generation through the first three postpartum days were observed. No apparent effects on reproductive outcome were noted. NOAEL can be considered to be 365 mg/kg/d for maternal and developmental toxicity.
 This study shows that N,N-dimethylaniline doesn’t lead to adverse effects on reproductive performance of the parental mice nor on the viability and weight of litters.

- Substance-tailored exposure driven testing [if applicable]: This element is not relevant here.

- Approaches in addition to above [if applicable]: not applicable

- Other reasons [if applicable]: not applicable


CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

Extended one-generation reproductive toxicity or Two-generation reproductive toxicity
As indicated in the IUCLID dossier of NMA, no specific studies are available for the assessment of the possible effect of N-methylaniline exposure on fertility. However, the repeated dose studies available by oral and inhalation route show no significant effects on reproductive organs of either male or female at concentrations that induce significant effects in the blood system and the target organ (spleen). For aniline, a similar conclusion can be drawn.

Regarding the developmental toxicity, the key value is derived from one-generation reproductive toxicity study with aniline [13] and is supported by another developmental study with aniline and N,N-dimethylaniline [5]. Considering the high rate of similarity in the mode of action between aniline and methylaniline, the value for NMA is copied from the study with aniline. A NOEL of 10 mg/kg/day was found for all embryotoxic, teratologic, and postnatal endpoints for the offspring.

Since aniline has proven to pass the placental barrier relatively easily [15], this is believed to be also true for NMA. Both substances are also easily absorbed by skin. Furthermore, the type of route of exposure does not affect the mode of action of these anilines. Hence, here the NOAEL recorded for the teratological potential of aniline can be safely used as an estimated value for N-methylaniline.

Another information could be pointed out from the 13-week study in rats with N,N-dimethylaniline [9]. Indeed, it is mentioned that “compound-related extramedullary hematopoiesis and hemosiderosis occurred in the kidney or testis of dosed rats”, meaning that the testis is targeted by this substance. This is a clue for a reproductive toxicity finding but it remains to be investigated and also, the extrapolation to NMA has to be clarified.

Overall, no interesting new information on NMA was found to fill the information gap regarding the extended one-generation reproductive toxicity or the two-generation reproductive toxicity. However, from the long-term repeated dose toxicity studies on aniline and N,N-dimethylaniline, no reproductive adverse effects were noted [3, 9]. Moreover, no fertility findings were identified for aniline or N,N-dimethylaniline in an one-generation reproductive study [12]. By extrapolating the results of the analogues to NMA, this allows to conclude that, based on these studies, there is no evidence of fertility adverse effects following treatment. Information related to developmental toxicity is already available in NMA dossier, from a read-across on aniline and N,N-dimethylaniline [5, 13].

Therefore, the argumentation here which aimed initially to justify the need for a test shows that an extended one-generation reproductive toxicity study or two-generation reproductive toxicity study might not be necessary to conduct, if ECHA judges that it is enough robust. Indeed, no evidence of fertility adverse effects were observed from the repeated dose toxicity studies of two analogues of NMA, i.e. aniline and N,N-dimethylaniline.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION: no

REFERENCES:
[1] Abe et al, “Embryonic development assay with Daphnia magna: application to toxicity of aniline derivatives.”, Chemosphere. 2001.
[2] ECHA, Guidance on Information Requirements and Chemical Safety Assessment, chap R.7a: endpoint specific guidance, https://echa.europa.eu/documents/10162/13632/information_requirements_r7a_en.pdf/e4a2a18f-a2bd-4a04-ac6d-0ea425b2567f
[3] ECHA, Registration dossier of aniline, https://echa.europa.eu/fr/registration-dossier/-/registered-dossier/15333/7/6/2/?documentUUID=e4de3da0-d7c6-4f71-b65d-7a28d98907d8
[4] Groth et al, “Toxicity studies in fertilized zebrafish eggs treated with N-methylamine, N,N-dimethylamine, 2-aminoethanol, isopropylamine, aniline, N-methylaniline, N,N-dimethylaniline, quinone, chloroacetaldehyde, or cyclohexanol.”, Bull Environ Contam Toxicol. 1993.
[5] Hardin et al, “Evaluation of 60 Chemicals in a Preliminary Developmental Toxicity Test.”, Teratog Carcinog Mutagen, 1987.
[9] National Toxicology Program, report no 360, https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr360.pdf, citing Abdo et al, “Subchronic (13-week) toxicity studies of N,N-dimethylaniline administered to Fischer 344 rats and B6C3F1 mice.”, J Toxicol Environ Health. 1990.
[12] Piccirillo et al, “Screening of Priority Chemicals for Reproductive Hazards.”, Borriston Laboratories, 1983 https://ntrl.ntis.gov/NTRL/
[13] Price et al, “Teratologic and postnatal evaluation of aniline hydrochloride in the Fischer 344 rat.”, Toxicol. Appl. Pharmacol., 1985.
[15] SCOEL, “Recommandation from the Scientific Committee on Occupational Exposure Limits for N-Methylaniline”, 2012, citing Treon et al, “The toxic properties of xylidine and monomethylaniline. I. The comparative toxicity of xylidine and monomethylaniline when administered orally or intravenously to animals or applied upon their skin”. J Ind Hyg Toxicol, 31:1-20, 1949.
[16] Sitarek et al, “Developmental toxicity of N-methylaniline following prenatal oral administration in rats.”, Int J Occup Med Environ Health, 2016.

Data source

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion