Reaction mass of Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate and Octadec-9-en-1-yl ammonium mono- and di-butylphosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity Weight of Evidence Assessment for EC 434-280-4

EC 434-280-4 has a robust toxicology data set but lacks reproductive and developmental toxicity data. EC 434-280-4 is a salt and is expected to dissociate in aqueous environments back to the starting materials. This is supported by:

1. The substance completely dissolves in aqueous media, which demonstrates that hydrogen bonding is occurring with the ionized dissociation products this is evident in the water solubility study for EC 434-280-4.

2. Analytical identification via aqueous HPLC/MS identifies independent peaks from the substance.

3. Comparable toxicology data between EC 434-280-4 and the starting materials, particularly (Z)-octadec-9-enylamine (CAS 112-90-3).

 

Therefore, the reproductive toxicity can be assessed by evaluating the starting materials. The starting materials and their available reproductive toxicity information are as follows:

1.(Z)-octadec-9-enylamine (CAS 112-90-3)is a primary alkylamine evaluated by Germany in a EU Risk Assessment Report. There is adequate data for developmental toxicity; for reproductive toxicity, there is adequate data for a similar primary alkyl amine. This includes sufficient data to use in a risk assessment.

2.Dibutyl hydrogen phosphate (CAS 107-66-4, AKA ‘DHP’) and phosphoric acid, butyl ester (CAS12788-93-1, AKA ‘BAP’)are alkyl phosphate esters. An OECD 422 study that tested a mixture of mono, di, and tri-butyl esters is available and summarized in an OECD SIAR.

3.Di-n-hexyl dithiophosphoric acid (78-64-8, AKA DHDPA)is an alky dithiophosphoric acid. Due to the corrosivity of alkyl dithiophosphoric acids, there is an understandable lack of repeat dose and reproductive toxicity data for these materials. Two substances that are zinc salts of a dithiophosphoric acid are used to assess the reproductive toxicity of the alkyl dithiophosphate functionality.

a. Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8) was tested in an OECD 421 study.

b. Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts (CAS 68457-79-4) was tested in an OECD 422 study.

Robust study summaries of the reproductive toxicity data for the starting materials are provided.Based on this existing reproductive toxicity data for the starting materials, none are classified as reproductive hazards. Therefore, EC 434-280-4 is not expected to cause reproductive or developmental toxicity. In addition, there were no impacts to the reproductive organs evaluated (males: testes and epididymides; females: ovaries) in the valid OECD 407 conducted with EC 434-280-4. Regarding the risk assessment, there is adequate information to extrapolate a point of departure for the reproductive endpoint.

The following weight of evidence assessment provides the following:

1. Justification for using starting materials to assess reproductive toxicity of EC 434-280-4 (refer to additional information)

2. Summary of the reproductive toxicity data for the starting materials, including values applied for the risk assessment of EC 434-280-4

(refer to additional information)

In conclusion, no additional animal testing is warranted as the reproductive toxicity is adequately assessed for EC 434-280-4 by evaluating the reproductive toxicity potential of the starting materials that are used to make EC 434-280-4. This approach is similar to scenario 1 in the ECHA Read-Across Assessment Framework (RAAF), which covers transformation to common products. However, in this case the transformation products themselves are assessed.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

Justification for using starting materials to assess reproductive toxicity of EC 434-280-4

EC 434-280-4 is made by the neutralization of DHP, BAP, and DHDPA with 4 equivalents of (Z)-octadec-9-enylamine.These salt species are expected dissociate in aqueous environments, including upon ingestion. It is reasonable to expect that the toxicology of EC 434-280-4 is dependent upon the biological active functional groups present, regardless of whether it is present as a salt or as the unreacted starting materials. This is supported by comparing the available data for EC 434-280-4 with that of the starting materials. The amount of each starting material can be found in the table below.

Amount of each starting material used to make EC 434-280-4.

Starting Material	Wt% in CAS EC 434-280-4	Identifier used in assessment
(Z)-octadec-9-enylamine	53%	None
Di-n-hexyl dithiophosphoric acid	30%	DHDPA
Dibutyl hydrogen phosphate	9%	DHP
Phosphoric acid, butyl ester	8%	BAP

Table 2 compares the toxicity of(Z)-octadec-9-enylamine and EC 434-280-4. (Z)-octadec-9-enylamine is the most relevant material for the comparison to demonstrate read across is appropriate as it has a strikingly similar hazard and toxicology data profile, including for repeat dose/target organ toxicity. The comparable toxicology outcome between these two demonstrates that evaluating the starting materials as the dissociation products are appropriate – i.e., the toxicology of EC 4834-280-4 is equivalent to the starting materials. In contrast, the hazards assigned to DHDPA, DHP, and BAP that are relevant for this assessment are limited to corrosivity, which is somewhat mitigated, as expected, by the salt and why this comparison focuses on (Z)-octadec-9-enylamine.

 

Note that a comparison of the physical and chemical (Ps&Cs) properties between EC 434-280-4 and the starting materials to support the weight of evidence/read across should be done with caution. The Ps&Cs of EC 434-280-4 are expected to differ between the starting materials but this does not exclude the validity of the weight of evidence assessment.

 

Following is an endpoint-by-endpoint comparison between (Z)-octadec-9-enylamine and EC 434-280-4 that summarizes the comparison in Table 2:

Aquatic toxicity: Both materials are very toxic to aquatic organisms (acute and chronic category 1), but(Z)-octadec-9-enylamine is approximately an order of magnitude more toxic. However, it is difficult to compare the aquatic toxicity directly as the studies with EC 434-280-4 are likely influenced by different water solubility of the reactants and starting materials. Therefore, the kinetics and how much is entering into the dosing solution may influence the outcome.

Acute toxicity/lethality: The LD50 values of(Z)-octadec-9-enylamine and EC 434-280-4 are comparable when the amount of (Z)-octadec-9-enylamine in EC 434-280-4 is accounted for.

Skin/eye irritation:(Z)-octadec-9-enylamine is corrosive whereas EC 434-280-4 is very irritating. This difference is likely due the salt species ameliorating some of the reactivity and irritation.

Genotoxicity: both materials were negative for genotoxicity.

Repeat dose toxicity: Administration of both (Z)-octadec-9-enylamine and EC 434-280-4 over 28-days resulted in the same or similar effects at somewhat similar doses. When taking into account studies with other primary alkylamines, the effects that are in common include the following:

-Mortalities at high doses

- Impaired health condition, uncoordinated movements/gait, general poor condition of the animals, reduction in food consumption

- Increased white blood cell, neutrophil, and monocyte counts

- Erosion of the GI leading to squamous/mucosal hyperplasia

- Atrophy of the thymus

- Histiocytic/macrophage infiltration and aggregation in the intestine and mesenteric lymph nodes

Comparison of the toxicity data for(Z)-octadec-9-enylamine and EC 434-280-4

Endpoint	EC 434-280-4	(Z)-octadec-9-enylamine (112-90-3)
Ps&Cs	EC 434-280-4	(Z)-octadec-9-enylamine (112-90-3)
Kow	4.7	7.5 (calculated) (EU RAR)
Water solubility	1.2 mg/L	Insoluble at 25C 0.07639 mg/L (calculated) (EU RAR)
Vapor pressure	5.564 X 10^-3Pa at 20C	0.005 hPa at 20C (EU RAR)
Aquatic Toxicity	EC 434-280-4	(Z)-octadec-9-enylamine (112-90-3)
Endpoint	Result	Result
Acute Toxicity to Fish (OECD 203)	96-hr LL50= 7.3 mg/L (nominal loading rates – WAF) 96-hr NEOL = 1.8 mg/L (WAF)	96 h LC50 = 0.11 mg/L (EU RAR and RAC)
Acute Toxicity to Invertebrates (OECD 202)	48-hr EL50= 0.15 mg/L (WAF) 48-hr NOEL = 0.090 mg/L (WAF)	48 h EC50 = 0.011 mg/L (EU RAR)
Acute Toxicity to Algae (OECD 201)	Cell density 96-hr EL50= 0.13 mg/L (WAF) Growth rate 96-hr EL50= 0.17 mg/L (WAF) NOEL = 0.050 mg/L (WAF)	72 hr EC50 = 0.083 mg/L (EU RAR)
Mammalian Toxicity	EC 434-280-4	(Z)-octadec-9-enylamine (112-90-3)
Endpoint	Result	Result
Acute Oral Toxicity (rats) (OECD 401)	LD50= 4570 mg/kg (males) LD50= 3770 mg/kg (females)	LD50 = 1689 mg/kg (RAC)
Acute Dermal Toxicity (rats) (OECD 402)	LD50> 2.0 g/kg	NDA (material is corrosive)
Primary Eye Irritation (rabbits) (OECD 405)	24-hr Draize score = 26/110 24-72 hour average scores: 0.6, 0.5, 2.4, 1.7 (opacity, iritis, conjunctival redness, chemosis)	Classified as corrosive to skin category 1B
Primary Skin Irritation (rabbits) (OECD 404)	PII = 6.6/8.0 Mean 24-72 hour erythema and edema scores: 3.8, 3.4	Classified as corrosive to skin category 1B
Skin Sensitization (guinea pigs - Buehler) (OECD 406)	Negative for sensitization	NDA
In Vitro Gene Mutation (OECD 471)	Not mutagenic	Not mutagenic (EU RAR)
In Vitro Chromosomal Aberration (OECD 473)	Not clastogenic	Not clastogenic (EU RAR)
Micronucleus Test in the Mouse (OECD 474)	Not genotoxic	Not genotoxic (EU RAR)
Repeated-Dose Toxicity (OECD 407) – oral gavage	NOAEL = 30 mg/kg bw/day LOAEL = 100 mg/kg bw/day   Summary of results:   300 mg/kg bw/day: ·        Deaths and moribund animals leading to sacrificing dose group by day 11 ·        Emaciation, hunched posture, lethargy, uncoordinated movements and general poor condition of the animals ·        Histiocytic/macrophage in small intestine and mesenteric lymph nodes, possibly representing phagocytosed test item. ·        Increased white blood cell, neutrophil, and monocyte counts ·        Squamous hyperplasia of forestomach epithelium ·        Increased lymphocytolysis and lymphoid atrophy in the thymus   100 mg/kg bw/day: ·        Reduced body weight gain and food intake (males only) ·        Increased white blood cell and neutrophil, counts ·        Significant increase in platelets (males) ·        Minimal to moderate histiocytosis of mesenteric lymph node ·        Foamy macrophages in small intestine	NOAEL: 3.25 mg/kg bw/day ((Z)-octadec-9-enylamine)   Summary of results:   50 mg/kg bw/day ((Z)-octadec-9-enylamine): ·        Impaired health condition (reduced body weight gain, impaired motility, uncoordinated gait) ·        Reduction in food consumption ·        Thymus atrophy ·        Mucosal hyperplasia ·        Histiocyte accumulation ·        Hemoconcentration and leucocytosis/neutrophilemia   150 mg/kg bw/day (primary alkylamines, e.g., tallow alkyl amines) ·        Mortalities ·        Erosion of gastrointestinal mucosa   General findings for primary alkylamines ·        Histiocytic granuloma in intestinal wall and mesenteric lymph nodes ·        Histiocytic hyperplasia in mesenteric lymph nodes and intestine   (EU RAR and RAC)

The similarity between(Z)-octadec-9-enylamine and EC 434-280-4 is further supported by the hazard classifications for each:

EC 434-280-4	(Z)-octadec-9-enylamine
Skin irritant – Category 2	Acute oral toxicity – category 4
Eye irritant – Category 2	Skin corrosion – category 1B
Target organ toxicity, repeated exposure – category 2	Target organ toxicity, single exposure (respiratory irritation) – category 3
Environment (Acute/Chronic) category 1	Target organ toxicity, repeated exposure – category 2
	Environment (Acute/ Chronic ) – category 1 (M10)

Regarding the remaining starting materials that EC 434-280-4 will disassociate to,DHDPA, DHP, and BAP are typically only classified as corrosive to skin. Regarding the repeat dose data for DHDPA, DHP, and BAP, effects observed are limited to those that occur from direct site-of-contact toxicity due to the corrosive/irritating properties of these materials. For example:

 

- The OECD 422 study with DHP/BAP resulted in a NOAEL of 30 mg/kg bw/day based on effects in the stomach (epithelial hyperplasia) and urinary bladder (epithelial hyperplasia) and was attributed to site of entry (stomach) or excretion (bladder). No other adverse effects or target organ toxicity was noted.

 

- The two repeat dose studies with the surrogate test material for the alkyl dithiophosphoric acid:

 

The OECD 421 study with CAS 4259-15-8 resulted in a NOAEL of 30 mg/kg bw/day. Parental toxicity occurred at doses of 100 and 200 mg/kg bw/day (mortality, clinical signs, gastric irritation – no other target organ related findings occurred).

The OECD 422 study with CAS 68457-79-4 resulted in a NOAEL of 160 mg/kg bw/day (highest dose) for systemic toxicity and 40 mg/kg bw/day for portal of entry effects, including epithelial hyperplasia, hyperkeratosis, and inflammation of the stomach.

2. Summary of the reproductive toxicity data for the starting materials, including values applied for the risk assessment of EC 434-280-4

The table below summarizes the reproductive toxicity NOAELs available for each starting material in EC 434-280-4. In addition, Table 3 contains the adjusted reproductive toxicity NOAEL based on additional correction factors. For the butyl phosphate esters, DHP and BAP, correction factors includes accounting for the amount of each present in the available reproductive toxicity study; based on the similarity between the two phosphate esters and the lack of reproductive toxicity, this adjustment is likely very conservative. The final correction factor is based on the amount of each starting material present in EC 434-280-4. 

PoD selection for reproductive/developmental toxicity

Starting Material in EC 434-280-4	Reproductive toxicity NOAEL	Basis	Amount in EC 434-280-4	Reproductive toxicity NOAEL/PoD for EC 434-280-4
(Z)-octadec-9-enylamine	12.5 mg/kg/day (fertility)	OECD 421 with tallow alkylamines (61790-33-8)	53%	23.6 mg/kg/day (= 12.5/0.53)
Di-n-hexyl dithiophosphoric acid	30 mg/kg/day (neonatal)	OECD 421 with CAS 4259-15-8	30%	100 mg/kg/day (= 30/0.3)
Dibutyl hydrogen phosphate	300 mg/kg/day (neonatal)	OECD 422 with DHP (107-66-4)   62.6% DHP present	9%	2086 mg/kg/day (= 300 x 0.626 / 0.09)
Phosphoric acid, butyl ester	300 mg/kg/day (neonatal)	OECD 422 with DHP (107-66-4)   18.3% of BAP present	8%	686 mg/kg/day (= 300 x 0.183 / 0.08)

The lowest NOAEL for reproductive toxicity is 23.6 mg/kg bw/day, which is based upon the reproductive toxicity for primary alkyl amines. Using the reproductive toxicity data for (Z)-octadec-9-enylamine for the risk assessment is consistent with the similar observations between this material and EC 434-280-4 in the repeat dose toxicity study. Following is a summary of the reproductive toxicity data for each starting material as well as a justification for why using these studies are applicable. Additional information is found in the Robust Study Summaries.

(Z)-octadec-9-enylamine (CAS 112-90-3)

 

A thorough and complete assessment was conducted by Germany in an EU Risk Assessment Report that was then used as the basis for a RAC opinion. The assessment was for multiple similar primary alkyl amines. Two developmental toxicity studies are available for (Z)-octadec-9-enylamine, one in rat and one in rabbit. There was no embryotoxicity or teratogenicity observed and the NOAEL was 30 mg/kg bw/day. An OECD 421 on tallow alkylamines (CAS 61790-33-8) was used to assess reproductive toxicity for the category. A NOAEL of 12.5 mg/kg bw/day was assigned based on findings of a lower fertility index and a lower conception rate at the next highest dose (50 mg/kg bw/day).

 

Dibutyl hydrogen phosphate (CAS 107-66-4, AKA ‘DHP’) and phosphoric acid, butyl ester (CAS12788-93-1, AKA ‘BAP’)

 

An OECD 422 study was conducted by MHW, Japan on dibutyl hydrogen phosphate (107-66-4). The test material was a mixture of mono, di, and tri-butyl phosphate ester. Therefore, this study is used to assess the reproductive toxicity of both DHP and BAP.

 

No study report is available, so this study is assigned a Klimisch code of 4. However, there is a Japan Chemicals Database Abstract available as well an OECD SIAR (by Japan) that would have evaluated the merits of this study and concluded on the outcome. The study resulted in a NOAEL of 1000 mg/kg bw/day for reproductive toxicity and 300 mg/kg bw/day for neonatal toxicity based on a high incidence of fatalities of pups in certain litters at or after birth.

 

Di-n-hexyl dithiophosphoric acid (78-64-8, AKA DHDPA)

 

Based on a comprehensive literature search, there is minimal information on dithiophosphate alkyl esters such as di-n-hexyl dithiophosphoric acid (for example, see the US EPA Screening-Level Hazard Characterization for the Dithiophosphate Alkyl Esters Category, December, 2009, which collected very little data on this category). This is consistent with the fact that these materials are highly corrosive to skin and would be difficult to test in repeat dose studies. However, there is sufficient reproductive data on zinc dialkyl dithiophosphate esters.

 

Zinc dialkyl dithiophophate (ZDDP) estersare complexes of zinc salts. They are expected to dissociate to the cationic zinc and the anionic di-alkyl dithiophosphoric acid, especially under the acidic conditions of the stomach. Therefore, reading across from the 2 ZDDPs with reproductive toxicity data is a reasonable approach to assessing the reproductive toxicity for the dialkyl dithiophosphoric acid functionality in EC 434-280-4.

 

The table below summarizes the chemical structures and Ps&Cs of DHDPA, ZDDPs, and the ZDDP dithiophosphoric acid hydrolysis products. Two ZDDPs are included to increase the robustness of the data set and to bracket the molecular weight (MW) of di-n-hexyl dithiophosphoric acid.

Summary and comparison of dithiophosphate alkyl ester and analog substances used in assessment

Chemical Name	CAS Number	Avergae MW		Water solubility (mg/L)	Kow	Vapour Pressure (mm Hg at 25C)
Dialkyl dithiophosphoric acids
Di-n-hexyl dithiophosphoric acid (DHDPA)   Purpose: target substance (starting material in EC 434-280-4)	78-64-8	298		1.2 0.067*	5.0 6.17*	0.000114*
Phosphorodithioic acid, mixed O,O-bis(isobutyl and pentyl) esters   Purpose: dissociation product of CAS 68457-79-4, a ZDDP used as an analog.	68516-01-8	256		2.454*	4.62*	0.00134*
Phosphoric acid, O,O-bis(2-ethylhexyl) ester   Purpose: dissociation product of CAS 4259-15-8, a ZDDP used as an analog.	5810-88-8	354		0.00088*	7.99*	0.0000019*
Zinc dialkyl dithiophosphate esters
Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts   Purpose: Data used to assess the reproductive toxicity potential of DHDPA	68457-79-4	576		1658**	0.69**	0.0000188**
Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate)   Purpose: Data used to assess the reproductive toxicity potential of DHDPA	4259-15-8	772		9.1**	3.59**	0.000032**

*Modeled using EPI Suite v4.1

** Measured data from REACH Registration

Full study reports and robust study summaries are available for both ZDDPs.

 

Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8) was tested in an OECD 421 study. The reproductive NOAEL was 100 mg/kg bw/day. Reproductive performance was unaffected by treatment at the 30 and 100 mg/kg/day dose levels. Slightly reduced fertility indices occurred at the highest dose of 200 mg/kg bw/day.

 

The NOAEL for neonatal toxicity was 30 mg/kg bw/day. Neonatal toxicity (mortality) in the F1 generation was observed at the 100 and 200 mg/kg/day dose levels. Neonatal toxicity was also noted at the 200 mg/kg/day dose level by clinical signs. No neonatal toxicity was observed at a dose level of 30 mg/kg/day

 

Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts (CAS 68457-79-4) was tested in an OECD 422 study. The NOAEL for reproductive and neonatal toxicity was 160 mg/kg bw/day, the highest dose tested. No test item related effects on reproductive performance or neonatal toxicity occurred.

 

For the risk assessment on DHDPA, the lowest NOAEL was chosen, which was the neonatal toxicity NOAEL from the OECD 421 study with CAS 4259-15-8.